Short interfering nucleic acid (siNA) molecules containing a 2′ internucleoside linkage

ABSTRACT

The present invention relates to RNAi molecules, and compositions thereof, comprising a 2′ internucleoside linkage connecting the nucleotide at position 1 and the nucleotide at position 2 at the 5′ end of the antisense strand. Specifically, the invention relates to single- and double-stranded short interfering nucleic acid (siNA) molecules that are capable of mediating RNA interference comprising 5′ modified nucleotides that comprise, among other potential modifications, a 2′ internucleoside linkage. The invention further relates to 5′ modified nucleotides used as reagents to generate the RNAi molecules of the invention and methods of using the disclosed RNAi molecules.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is the U.S. National Phase Application under 35U.S.C. § 371 of International Application No. PCT/US2014/017260, filedFeb. 20, 2014, which claims priority to U.S. Provisional Application No.61/767,837, filed Feb. 22, 2013, which applications are herebyincorporated by reference herein in their entireties.

REFERENCE TO SEQUENCE LISTING

The sequence listing submitted via EFS-Web, in compliance with 37 CFR §1.52(e)(5), is incorporated herein by reference. The sequence listingtext file submitted via EFS contains the file A2038-7218US—SequenceListing.txt, was created on Oct. 19, 2015, and is 115,716 bytes in size.

BACKGROUND OF THE INVENTION

RNA interference (RNAi) is an evolutionarily conserved cellularmechanism of post-transcriptional gene silencing found in fungi, plantsand animals that uses small RNA molecules to inhibit gene expression ina sequence-specific manner. RNAi is controlled by the RNA-inducedsilencing complex (RISC) that is initiated by short double-stranded RNAmolecules in a cell's cytoplasm. The short double-stranded RNA interactswith Argonaute 2 (Ago2), the catalytic component of RISC, which cleavestarget mRNA that is complementary to the bound RNA. One of the two RNAstrands, known as the guide strand, binds the Ago2 protein and directsgene silencing, while the other strand, known as the passenger strand,is degraded during RISC activation. See, for example, Zamore and Haley,2005, Science, 309:1519-1524; Vaughn and Martienssen, 2005, Science,309:1525-1526; Zamore et al., 2000, Cell, 101:25-33; Bass, 2001, Nature,411:428-429; and, Elbashir et al., 2001, Nature, 411:494-498.Single-stranded short interfering RNA has also been shown to bind Ago2and support cleavage activity (see, e.g., Lima et al., 2012, Cell150:883-894). Importantly, the activity of single-stranded RNAimolecules should not be confused with that of single-stranded antisenseRNA that inhibits translation of a complementary RNA in a stoichiometricfashion by base pairing to it, physically obstructing the translationmachinery.

The RNAi machinery can be harnessed to destruct any mRNA of a knownsequence. This allows for suppression (knock-down) of any gene fromwhich it was generated, consequently preventing the synthesis of thetarget protein. Modulation of gene expression through an RNAi mechanismcan be used to modulate therapeutically relevant biochemical pathways,including ones which are not accessible through traditional smallmolecule control. RNAi has also become a very important tool for targetvalidation in the pharmaceutical industry.

Chemical modification of nucleotides incorporated into RNAi moleculesleads to improved physical and biological properties, such as nucleasestability (see, e.g., Damha et al., 2008, Drug Discovery Today,13:842-855), reduced immune stimulation (see, e.g., Sioud, 2006, TRENDSin Molecular Medicine, 12:167-176), enhanced binding (see, e.g., Koller,E. et al., 2006, Nucleic Acid Research, 34:4467-4476), and enhancedlipophilic character to improve cellular uptake and delivery to thecytoplasm. Thus, chemical modifications have the potential to increasepotency of RNA compounds, allowing lower doses of administration,reducing the potential for toxicity, and decreasing overall cost oftherapy.

While the sugar-phosphate backbone of most DNAs and RNAs are comprisedof 3′-5′ internucleoside phosphodiester linkages, the physiochemical andbiochemical properties of 2′-5′ linked ribonucleotides have beenstudied. Although not used for biological information storage, 2′-5′linked oligoribonucleotides support Watson-Crick base pairing and areformed naturally during intron splicing and in interferon treated cells(see, e.g., Jim et al., 1993, Proc. Natl. Acad. Sci. USA,90:10568-10572; Sawai et al., 1996, Biopolymers, 39:173-182; Premraj etal., 2002, Biophysical Chemistry, 95:253-272; Johnston and Torrence(1984) in Interferons: Mechanism of Production and Action, Vol. 3(Friedman, R. M., Ed.), pp. 189-298, Elsevier, Amsterdam). There hasbeen interest in using 2′-5′ linked oligoribonucleotides in antisenseRNA applications as they exhibit the tendency to selectively hybridizewith their RNA complements, rather than DNA complements (see, e.g.,Hashimoto and Switcher, 1992, J. Am. Chem. Soc., 114:6255-6256;Dougherty et al., 1992, J. Am. Chem. Soc., 114:6265-6255), and displayimproved resistance toward several types of nucleases (see, e.g., Alluland Hoke, 1995, Antisense Res. Develop., 5:3-11; Kandimalla et al.,1997, Nucl. Acids Res., 25:370-378; Prakash et al., 1999, Bioorg. Med.Chem. Lett., 9:2515-2520). However, there has been only limited study ofthe impact of 2′-5′ linked ribonucleotides within RNAi oligonucleotideson the ability for such 2′-5′ linked oligoribonucleotides toappropriately and efficiently degrade target gene expression through anAgo2-mediated RNAi pathway.

Prakish et al. (2006, Bioorg. Med. Chem. Lett 16:3238-3240) reported onthe activity in mammalian cells of siRNA duplexes that have 2′-5′ linkednucleotides. Results showed that an siRNA duplex comprising a 2′-5′linked antisense strand and a 3′-5′ linked sense strand was not activein inhibiting mRNA expression, while an siRNA duplex with the reversecomposition (i.e., a 3′-5′ linked antisense strand and a 2′-5′ linkedsense strand) was active. They concluded that 2′-5′ linkages aretolerated in the sense strand of siRNA duplexes but not in the antisensestrand. Since the 5′-end of the antisense strand, in particular, isimportant for loading siRNA into RISC, positioning nucleation with mRNAand subsequent cleavage, the authors provide that it is likely crucialfor the 5′ end of the antisense strand to adopt correct geometry inorder to appropriately interact with RISC. They conclude that 2′-5′internucleoside linkages at the 5′ end of the antisense strand, thus,may not be capable of adopting the proper conformation to support thatinteraction.

PCT International application serial no. PCT/US2011/033961, published asWO 2011/139699 on Apr. 26, 2011, discloses 5′ modified nucleosides andoligomeric compounds incorporating the modified nucleosides. The 5′modified nucleosides disclosed are preferably located at the 5′ terminusof an oligonucleotide and have modifications at the 5′ carbon of thesugar moiety of the nucleoside and, optionally, additional modificationsat the 2′ carbon. The 5′ modified nucleosides disclosed inPCT/US2011/033961 are linked to an adjacent nucleoside by a traditional3′-5′ internucleoside linkage.

SUMMARY OF THE INVENTION

The instant disclosure provides novel single- or double-stranded smallnucleic acid molecules capable of mediating RNA interference comprisingan antisense strand that is complementary to a nucleic acid target andhaving a modified nucleotide at the 5′ end comprising a 2′internucleoside linkage (e.g., 2′-5′ internucleoside linkage). Thesingle- or double stranded small nucleic acid molecules of the inventionare more specifically referred to herein as short interfering nucleicacid (siNA) molecules, and the modified nucleotide at the 5′ end of theantisense strand of said molecules is referred to herein as a 5′modified nucleotide. The 5′ modified nucleotide makes up position 1 atthe 5′ end of the antisense strand of the siNA molecules of theinvention (i.e., the first nucleotide of the 5′ end of the strand).Novel 5′ modified nucleotides for use as reagents to generate the siNAmolecules disclosed herein are also part of the instant invention. Thepresent invention further includes methods of modulating (e.g.,inhibiting) the expression of genes, in vitro or in vivo, using siNAmolecules disclosed herein and compositions thereof. Some embodiments ofthe siNA molecules of the invention are shown herein to display improvedactivity (see Examples, infra).

In one aspect, the present invention relates to short-interferingnucleic acid (siNA) molecules that are capable of mediating RNAinterference (RNAi) and comprise an antisense strand having a 5′modified nucleotide. The antisense strand of the siNA molecules of theinvention is either partially or completely complementary to a nucleicacid target. The siNA molecules of the invention can be single- ordouble-stranded small nucleic acid molecules and can take differentoligonucleotide forms, including but not limited to short interferingRNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA) and shorthairpin RNA (shRNA) molecules. The 5′ modified nucleotide at nucleotideposition 1 of the antisense strand of the siNA molecules of theinvention is linked to a nucleotide at position 2 of the strand througha 2′ internucleoside linkage and may contain a modified 5′ cap (i.e.,other than a 5′ phosphate cap). In particular, the short interferingnucleic acid (siNA) molecules of the invention comprise an antisensestrand having a 5′ modified nucleotide having the structure of FormulaII:

wherein:

A is —OC(R³)₂—, —C(R³)₂O—, —C(R³)₂—, —C(R³)₂C(R³)₂— or —CR³═CR³—;

B is any heterocyclic base moiety;

D¹ and D^(1′) are independently selected from hydroxyl, —OR⁴, —SR⁴, or—N(R⁴)₂;

E is O, S, —NR⁵, —N—N(R⁴)₂ or —N—OR⁴;

J is an internucleoside linking group linking the 5′ modified nucleotideof Formula II to the sugar moiety of an adjacent nucleotide of the siNAmolecule;

R¹ and R^(1′) are independently selected from H, hydroxyl, halogen, C₁₋₆alkyl, —OR⁶, —N(R⁶)₂, or together form ═O or ═CH₂;

R² is H, C₁₋₆ alkyl or C₂₋₆ alkenyl;

R³ and R⁵ are independently selected from H, hydroxyl, halogen, C₁₋₁₀alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, aryl,

wherein R′ is selected from H or C₁₋₄ alkyl (which is optionallysubstituted with one to three substituents independently selected from—SR¹⁰, aryl, heteroaryl, amino, hydroxyl, oxo or —NH—C═(NH)NH₂, whereinthe aryl and heteroaryl are optionally substituted with hydroxyl), andR″ is selected from H, C₁₋₁₈ alkyl or aryl;

R⁴ is independently selected from H, C₁₋₁₀ alkyl, C₂₋₆ alkenyl, C₂₋₆alkynyl, aryl,

wherein R′ is selected from H or C₁₋₄ alkyl (which is optionallysubstituted with one to three substituents independently selected from—SR¹¹, aryl, heteroaryl, amino, hydroxyl, oxo or —NH—C═(NH)NH₂, whereinthe aryl and heteroaryl are optionally substituted with hydroxyl), andR″ is selected from H, C₁₋₁₈ alkyl or aryl;

R⁶ is independently selected from H, C₁₋₆ alkyl (which is optionallysubstituted with —OR⁷, —SR⁷, —N(R⁸)₂, or (═O)—NR⁹ or from one to threehalogen), C₂₋₆ alkenyl, C₂₋₆ alkynyl, aryl,

wherein R′ is selected from H or C₁₋₄ alkyl (which is optionallysubstituted with one to three substituents independently selected from—SR¹⁰, aryl, heteroaryl, amino, hydroxyl, oxo, —NH—C═(NH)NH₂, whereinthe aryl and heteroaryl are optionally substituted with hydroxyl), andR″ is selected from H, C₁₋₁₈ alkyl or aryl;

R⁷ is methyl, —CF₃, —N(R⁸)₂ or —CH₂—N(R⁸)₂;

R⁸ is independently selected from H or C₁₋₆ alkyl;

R⁹ is (R⁸)₂, —R⁸—(CH₂)₂—N(R⁸)₂ or —R⁸—C(═NR⁸)[N(R⁸)₂]; and,

R¹⁰ is H or C₁₋₄ alkyl.

The siNA molecules of the invention can be single-stranded ordouble-stranded oligonucleotide molecules. The oligonucleotide moleculesof the invention may inhibit gene expression in a cell or animal via anRNA interference (RNAi) mechanism.

In one aspect, the invention provides single-stranded short interferingnucleic acid (siNA) molecules, wherein the single oligonucleotide strandcomprises a sequence that is complementary to at least part of a nucleicacid target sequence associated with gene expression. For purposes ofthis disclosure, the single strand of a single-stranded siNA molecule ofthe invention is referred to as the antisense strand.

In another aspect, the invention provides double-stranded shortinterfering nucleic acid (siNA) molecules, wherein a double-strandedsiNA molecule comprises a sense and an antisense strand. The antisensestrand comprises a sequence that is complementary to at least part of anucleic acid target sequence associated with gene expression, and thesense strand is complementary to at least part of the antisense strand.The double-stranded siNA molecules of the invention can be symmetric orasymmetric.

In certain embodiments, the siNA molecules of the invention comprise anantisense strand that is complementary to a portion of a target nucleicacid sequence, wherein the target nucleic acid is selected from: atarget mRNA, a target pre-mRNA, a target microRNA, and a targetnon-coding RNA. In certain embodiments, an siNA molecule of theinvention is a microRNA mimetic.

In certain embodiments, the siNA molecules of the invention comprise anantisense strand having at least 15 nucleotides having sequencecomplementarity to a target nucleic acid sequence. In certainembodiments, the antisense strand of an siNA molecule of the inventionis about 15 to 30 nucleotides in length. In other embodiments, adouble-stranded siNA molecule of the invention comprises a sense strandand an antisense strand, wherein each strand is independently about 15to 30 nucleotides in length.

In certain embodiments, the siNA molecules of the invention furthercomprise one or more additional nucleotides in either one or bothstrands of the molecule that are chemically-modified. Modificationsinclude nucleic acid sugar modifications, base modifications, backbone(internucleoside linkage) modifications, non-nucleotide modifications,and/or any combination thereof. In certain embodiments, the siNAmolecules of the invention comprise one or more modified internucleosidelinking groups. In certain embodiments, each internucleoside linkinggroup is, independently, a phosphodiester or phosphorothioate linkinggroup. In certain embodiments, any one or more additionalchemically-modified nucleotides in the antisense strand of either asingle- or double-stranded siNA of the invention does not have a 2′-5′internucleoside linkage. In certain embodiments, the antisense strand ofa single- or double-stranded siNA of the invention may contain up to 9additional chemically-modified nucleotides with 2′-5′ internucleosidelinkages.

In certain embodiments, the double-stranded siNA molecules of theinvention have 3′ overhangs of one, two, three or four nucleotide(s) onone or both of the strands. In other embodiments, the double-strandedsiNA molecules lack overhangs (i.e., have blunt ends).

In some embodiments, the siNA molecules of the invention have one ormore terminal caps (also referred to herein as “caps”). Forsingle-stranded siNA molecules of the invention, a cap may be present atthe 3′-terminus (3′-cap). For double-stranded siNA molecules of theinvention, a cap may be present at the 3′-terminus (3′-cap) of theantisense strand (guide strand), at the 5′-terminus (5′-cap) of thesense strand (passenger strand), and/or at 3′-terminus (3′-cap) of thesense strand (passenger strand).

The present invention further provides compositions comprising the siNAmolecules described herein with, optionally, a pharmaceuticallyacceptable carrier or diluent. The administration of the composition canbe carried out by known methods, wherein the nucleic acid is introducedinto a desired target cell in vitro or in vivo.

The molecules and compositions of the present invention have utilityover a broad range of applications related to modulating geneexpression, including potential therapeutic applications. Thus, oneaspect of this invention relates to the use of the molecules andcompositions of the invention to inhibit gene expression in a cell viaan RNAi mechanism. Methods comprise contacting a cell with a molecule orcomposition of the invention. In certain embodiments, such methodsfurther comprise detecting RNAi activity. Detection and/or measuring ofRNAi gene silencing activity may be direct or indirect.

Another aspect of this invention relates to administering the moleculesand compositions of the invention to a subject (e.g., an animal). Inthis aspect of the invention, the molecules and compositions have thepotential use of treating said subject, such as a human, who issuffering from a condition (such as cancer) which is mediated by theaction, or by the loss of action, of a target nucleic acid or protein.In certain embodiments, the invention provides use of an siNA moleculeof the invention for the manufacture of a medicament for the potentialtreatment of a disease by inhibiting gene expression.

The instant disclosure further provides novel 5′ modified nucleotidesthat can be used to generate a single- or double-stranded siNA moleculeas disclosed herein. When incorporated into an siNA molecule of theinvention, the novel modified nucleotides, generally referred to hereinas 5′ modified nucleotides, make up position 1 at the 5′ end of theantisense strand of the siNA molecule (i.e., the first nucleotide of the5′ end of the antisense strand), are linked to a nucleotide at position2 of the strand through a 2′ internucleoside linkage, and may contain amodified 5′ cap (i.e., other than a 5′ phosphate cap). In particular,the instant invention features 5′ modified nucleotides having thestructure of Formula I:

wherein:

A is —C(R³)₂—, —C(R³)₂C(R³)₂— or —CR³═CR³—;

B is any heterocyclic base moiety;

D¹ and D^(1′) are independently selected from hydroxyl, —OR⁴, —SR⁴, or—N(R⁴)₂;

E and E′ are independently selected from O, S, —NR⁵, —N—N(R⁴)₂ or—N—OR⁴;

G¹ is hydroxyl or —OR⁶;

G^(1′) is hydroxyl, —OR⁶ or —N(R⁶)₂;

R¹ and R^(1′) are independently selected from H, hydroxyl, halogen, C₁₋₆alkyl, —OR⁷, —N(R⁷)₂, or together form ═O or ═CH₂;

R² is H, C₁₋₆ alkyl or C₂₋₆ alkenyl;

R³ and R⁵ are independently selected from H, hydroxyl, halogen, C₁₋₁₀alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, aryl,

wherein R′ is selected from H or C₁₋₄ alkyl (which is optionallysubstituted with one to three substituents independently selected from—SR¹¹, aryl, heteroaryl, amino, hydroxyl, oxo or —NH—C═(NH)NH₂, whereinthe aryl and heteroaryl are optionally substituted with hydroxyl), andR″ is selected from H, C₁₋₁₈ alkyl or aryl;

R⁴ is independently selected from H, C₁₋₁₀ alkyl, C₂₋₆ alkenyl, C₂₋₆alkynyl, aryl,

wherein R′ is selected from H or C₁₋₄ alkyl (which is optionallysubstituted with one to three substituents independently selected from—SR¹¹, aryl, heteroaryl, amino, hydroxyl, oxo or —NH—C═(NH)NH₂, whereinthe aryl and heteroaryl are optionally substituted with hydroxyl), andR″ is selected from H, C₁₋₁₈ alkyl or aryl;

R⁶ is independently C₁₋₆ alkyl, optionally substituted on the terminalcarbon atom with cyano or a protecting group;

R⁷ is independently selected from H, C₁₋₆ alkyl (which is optionallysubstituted with —OR⁸, —SR⁸, —N(R⁹)₂, (═O)—NR¹⁰ or from one to threehalogen), C₂₋₆ alkenyl, C₂₋₆ alkynyl, aryl,

wherein R′ is selected from H or C₁₋₄ alkyl (which is optionallysubstituted with one to three substituents independently selected from—SR¹¹, aryl, heteroaryl, amino, hydroxyl, oxo, —NH—C═(NH)NH₂, whereinthe aryl and heteroaryl are optionally substituted with hydroxyl), andR″ is selected from H, C₁₋₁₈ alkyl or aryl;

R⁸ is methyl, —CF₃, —N(R⁹)₂ or —CH₂—N(R⁹)₂;

R⁹ is independently selected from H or C₁₋₆ alkyl;

R¹⁰ is (R⁹)₂, —R⁹—(CH₂)₂—N(R⁹)₂ or —R⁹—C(═NR⁹)[N(R⁹)₂];

R¹¹ is H or C₁₋₄ alkyl; and,

r is 0 or 1.

These and other aspects of the invention will be apparent upon referenceto the following Detailed Description and attached figures. Moreover, itis contemplated that any method or composition described herein can beimplemented with respect to any other method or composition describedherein and that different embodiments may be combined.

Additionally, patents, patent applications and other documents are citedthroughout the specification to describe and more specifically set forthvarious aspects of this invention. Each of these references cited hereinis hereby incorporated by reference in its entirety, including thedrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 compares knock-down activity (ddCT) of a subset ofsingle-stranded siNA molecules described in Table 2 at differingconcentrations (100 nM, 10 nM, 1 nM and 0.1 nM). The knock-down activitywas screened in Hepa1-6 cells transfected with RNAiMax (see Example 6).FIG. 1A compares the activity of single-stranded siNA molecules having5′-position 1 nucleotides with regular 3′-5′ internucleoside linkages(BM, BMs, dT, and dTs) to the activity of siNA molecules having5′-position 1 nucleotides with 2′-5′ internucleoside linkages (3dX and3dXs, where X is T, A, C or G). FIG. 1B compares the activity ofsingle-stranded siNA molecules, each having a 2′-5′ internucleosidelinkage at position 1 and various additional structural changes at thatposition.

FIG. 2A compares knock-down activity (ddCT) of the single-stranded siNAmolecules described in Table 8 at differing concentrations (100 nM, 10nM, 1 nM and 0.1 nM). The knock-down activity was screened in Hepa1-6cells transfected with RNAiMax (see Examples 6 and 7). The siNAmolecules shown in this figure each contain internal nucleotides with2′-5′ internucleoside linkages.

FIG. 2B compares knock-down activity (ddCT) of the single-stranded siNAmolecules described in Table 10 at differing concentrations (100 nM, 10nM, 1 nM and 0.1 nM). The knock-down activity was screened in Hepa1-6cells transfected with RNAiMax (see Examples 6 and 8). The siNAmolecules shown in this figure each contain a 2′-5′ internucleosidelinkage at position 1 and multiple phosphorothioate linkages.

DETAILED DESCRIPTION OF THE INVENTION A. Terms and Definitions

The following terminology and definitions apply as used in the presentapplication.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural referents unless the contentclearly dictates otherwise. Thus, for example, reference to “a cell”includes a combination of two or more cells, and the like.

Any concentration range, percentage range, ratio range or integer rangeis to be understood to include the value of any integer within therecited range, and when appropriate, fractions thereof (such as onetenth and one hundredth of an integer), unless otherwise indicated.

“About” or “approximately,” as used herein, in reference to a number aregenerally taken to include numbers that fall within a range of 5% ineither direction (greater than or less than) of the number unlessotherwise stated or otherwise evident from the context (except wheresuch number would exceed 100% of a possible value). Where ranges arestated, the endpoints are included within the range unless otherwisestated or otherwise evident from the context.

The phrases “2′-modified nucleotide,” “2′-substituted nucleotide” or anucleotide having a modification at the “2′-position” of the sugarmoiety, as used herein, generally refer to nucleotides comprising asubstituent at the 2′ carbon position of the sugar component that isother than H or OH. 2′-modified nucleotides include, but are not limitedto, bicyclic nucleotides wherein the bridge connecting two carbon atomsof the sugar ring connects the 2′ carbon and another carbon of the sugarring; and nucleotides with non-bridging 2′substituents, such as allyl,amino, azido, thio, O-allyl, OC₁₋₁₀ alkyl, —OCF3, O—(CH₂)₂—O—CH₃,2′-O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(R_(m))(R_(n)), orO—CH₂—C(═O)—N(R_(m))(R_(n)), where each R_(m) and R_(n) is,independently, H or substituted or unsubstituted C₁₋₁₀ alkyl.2′-modified nucleotides may further comprise other modifications, forexample at other positions of the sugar and/or at the nucleobase. Thephrases “3′-modified nucleotide,” “3′-substituted nucleotide” or anucleotide having a modification at the “3′-position” of the sugarmoiety generally refers to a nucleotide comprising a modification,including a substituent, at the 3′ carbon position of the sugarcomponent.

The term “abasic” as used herein refers to its meaning as is generallyaccepted in the art. The term generally refers to sugar moieties lackinga nucleobase or having a hydrogen atom (H) or other non-nucleobasechemical groups in place of a nucleobase at the 1′ position of the sugarmoiety, see for example Adamic et al., U.S. Pat. No. 5,998,203. In oneembodiment, an siNA molecule of the invention may contain an abasicmoiety, wherein the abasic moiety is ribose, deoxyribose, ordideoxyribose sugar.

The term “acyclic nucleotide” as used herein refers to its meaning as isgenerally accepted in the art. The term generally refers to anynucleotide having an acyclic ribose sugar, for example where any of theribose carbon/carbon or carbon/oxygen bonds are independently or incombination absent from the nucleotide.

If no number of carbon atoms is specified, the term “alkyl” refers to asaturated aliphatic hydrocarbon group, branched or straight-chain,containing from 1 to 10 carbon atoms. An alkyl group can have a specificnumber of carbon atoms. For example, C₁-C₁₀, as in “C₁-C₁₀ alkyl” or“C₁₋₁₀ alkyl,” is defined to include groups having 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 carbons in a linear or branched arrangement. For example,“C₁-C₁₀ alkyl” specifically includes methyl, ethyl, n-propyl, i-propyl,n-butyl, t-butyl, i-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl,and so on. The term “cycloalkyl” means a monocyclic saturated aliphatichydrocarbon group having the specified number of carbon atoms. Forexample, “cycloalkyl” includes cyclopropyl, methyl-cyclopropyl,2,2-dimethyl-cyclobutyl, 2-ethyl-cyclopentyl, cyclohexyl, and so on.

If no number of carbon atoms is specified, the term “alkenyl” refers toa non-aromatic hydrocarbon radical, straight or branched, containingfrom 2 to 10 carbon atoms and at least 1 carbon-to-carbon double bond.Preferably, one carbon-to-carbon double bond is present, and up to 4non-aromatic carbon-carbon double bonds may be present. Thus, “C₂-C₆alkenyl” or “C₂₋₆ alkenyl” means an alkenyl radical having from 2 to 6carbon atoms. Alkenyl groups include ethenyl, propenyl, butenyl andcyclohexenyl. The straight, branched or cyclic portion of the alkenylgroup may contain double bonds and may be substituted if a substitutedalkenyl group is indicated. The term “cycloalkenyl” means a monocyclichydrocarbon group having the specified number of carbon atoms and atleast one point of internal unsaturation with a carbon-to-carbon doublebond.

The term “alkynyl” refers to a hydrocarbon radical, straight orbranched, containing from 2 to 10 carbon atoms, unless otherwisespecified, and containing at least one carbon-to-carbon triple bond. Upto 3 carbon-carbon triple bonds may be present. Thus, “C₂-C₆ alkynyl” or“C₂₋₆ alkynyl” means an alkynyl radical having from 2 to 6 carbon atoms.Alkynyl groups include ethynyl, propynyl and butynyl. The straight orbranched portion of the alkynyl group may contain triple bonds and maybe substituted if a substituted alkynyl group is indicated.

The term “amino” refers to the group (—NH₂).

The term “antisense region” as used herein refers to its meaning as isgenerally accepted in the art. With reference to exemplary nucleic acidmolecules of the invention, the term refers to a nucleotide sequence ofan siNA molecule having complementarity to a target nucleic acidsequence. In addition, the antisense region of an siNA molecule canoptionally comprise a nucleic acid sequence having complementarity to asense region of the siNA molecule. In one embodiment, the antisenseregion of an siNA molecule is referred to as the antisense strand orguide strand.

As used herein, “aryl” is intended to mean any stable monocyclic orbicyclic carbon ring of up to 7 atoms in each ring, wherein at least onering is aromatic. Examples of such aryl elements include phenyl,naphthyl, tetrahydronaphthyl, indanyl and biphenyl. In cases where thearyl substituent is bicyclic and one ring is non-aromatic, it isunderstood that attachment is via the aromatic ring.

The term “biodegradable” as used herein refers to its meaning as isgenerally accepted in the art. The term generally refers to degradationin a biological system, for example, enzymatic degradation or chemicaldegradation.

The term “biodegradable linker” as used herein refers to its meaning asis generally accepted in the art. With reference to nucleic acidmolecules of the invention, the term refers to a linker molecule that isdesigned to connect one molecule to another molecule, and which issusceptible to degradation in a biological system. The linker can be anucleic acid or a non-nucleic acid-based linker. For example, abiodegradable linker can be used to attach a ligand or biologicallyactive molecule to an siNA molecule of the invention. Alternately, abiodegradable linker can be used to connect the sense and antisensestrands of an siNA molecule of the invention. A biodegradable linker isdesigned such that its stability can be modulated for a particularpurpose, such as delivery to a particular tissue or cell type. Thestability of a nucleic acid-based biodegradable linker molecule can bemodulated by using various chemistries, for example combinations ofribonucleotides, deoxyribonucleotides, and chemically-modifiednucleotides, such as 2′-O-methyl, 2′-fluoro, 2′-amino, 2′-O-amino,2′-C-allyl, 2′-O-allyl, and other 2′-modified or base modifiednucleotides. A biodegradable nucleic acid linker molecule can be adimer, trimer, tetramer or longer nucleic acid molecule, for example, anoligonucleotide of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, or 20 nucleotides in length, or can comprise a singlenucleotide with a phosphorus-based linkage, for example, aphosphoramidite or phosphodiester linkage. A biodegradable nucleic acidlinker molecule can also comprise nucleic acid backbone, nucleic acidsugar, or nucleic acid base modifications.

The term “biologically active molecule” as used herein refers to itsmeaning as is generally accepted in the art. With reference to exemplarynucleic acid molecules of the invention, the term refers to compounds ormolecules that are capable of eliciting or modifying a biologicalresponse in a system and/or are capable of modulating thepharmacokinetics and/or pharmacodynamics of other biologically activemolecules. Examples of biologically active molecules, include siNAmolecules alone or in combination with other molecules including, butnot limited to, therapeutically active molecules such as antibodies,cholesterol, hormones, antivirals, peptides, proteins,chemotherapeutics, small molecules, vitamins, co-factors, nucleosides,nucleotides, oligonucleotides, enzymatic nucleic acids, antisensenucleic acids, triplex forming oligonucleotides, polyamines, polyamides,polyethylene glycol, other polyethers, 2-5A chimeras, siNA, dsRNA,allozymes, aptamers, decoys and analogs thereof.

The term “biological system” as used herein refers to its meaning as isgenerally accepted in the art. The term generally refers to material, ina purified or unpurified form, from biological sources including, butnot limited to, human or animal, wherein the system comprises thecomponents required for RNAi activity. Thus, the phrase includes, forexample, a cell, tissue, subject, or organism, or extract thereof. Theterm also includes reconstituted material from a biological source.

The term “blunt end” as used herein refers to its meaning as isgenerally accepted in the art. With reference to nucleic acid moleculesof the invention, the term refers to termini of a double-stranded siNAmolecule having no overhanging nucleotides. For example, the two strandsof a double-stranded siNA molecule having blunt ends align with eachother with matched base-pairs without overhanging nucleotides at thetermini. An siNA duplex molecule of the invention can comprise bluntends at one or both termini of the duplex, such as termini located atthe 5′-end of the antisense strand, the 5′-end of the sense strand, orboth termini of the duplex.

The term “cap” (also referred to herein as “terminal cap”) as usedherein refers to its meaning as is generally accepted in the art. Withreference to exemplary nucleic acid molecules of the invention, the termrefers to a moiety, which can be a chemically-modified nucleotide or anon-nucleotide, incorporated at one or more termini of the nucleic acidmolecules of the invention. These terminal modifications may protect thenucleic acid molecule from exonuclease degradation and may help indelivery and/or localization of the nucleic acid molecule within a cell.The cap can be present at a 5′-terminus (5′-cap) or 3′-terminus (3′-cap)of a strand of the nucleic acid molecules of the invention, or can bepresent on both termini. For example, a cap can be present at the5′-end, 3′-end and/or 5′ and 3′-ends of the sense strand of a nucleicacid molecule of the invention. Additionally, a cap can be present atthe 3′-end of the antisense strand of a nucleic acid molecule of theinvention. In non-limiting examples, a 5′-cap includes, but is notlimited to, LNA; glyceryl; inverted deoxy abasic residue (moiety);4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide,4′-thio nucleotide; carbocyclic nucleotide; 1,5-anhydrohexitolnucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide;phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic3′,4′-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic3,5-dihydroxypentyl nucleotide; 3′-3′-inverted nucleotide moiety;3′-3′-inverted abasic moiety; 3′-2′-inverted nucleotide moiety;3′-2′-inverted abasic moiety; 1,4-butanediol phosphate;3′-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3′-phosphate;3′-phosphorothioate; phosphorodithioate; or bridging or non-bridgingmethylphosphonate moiety. Non-limiting examples of a 3′-cap include, butare not limited to, LNA; glyceryl; inverted deoxy abasic residue(moiety); 4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl)nucleotide; 4′-thio nucleotide; carbocyclic nucleotide; 5′-amino-alkylphosphate; 1,3-diamino-2-propyl phosphate; 3-aminopropyl phosphate;6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropylphosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide;alpha-nucleotide; modified base nucleotide; phosphorodithioate;threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide;3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide,5′-5′-inverted nucleotide moiety; 5′-5′-inverted abasic moiety;5′-phosphoramidate; 5′-phosphorothioate; 1,4-butanediol phosphate;5′-amino; bridging and/or non-bridging 5′-phosphoramidate;phosphorothioate and/or phosphorodithioate; bridging or non-bridgingmethylphosphonate; and 5′-mercapto moieties (for more details seeBeaucage and Iyer, 1993, Tetrahedron 49, 1925; incorporated by referenceherein). In one embodiment, siNA molecules of the present inventioncontain a vinyl phosphate 5′ terminal cap, wherein carbon 5 of the sugarring contains the following substituent (═CH)—P(═O)(OH)₂.

The term “cell” as used herein refers to its meaning as is generallyaccepted in the art. With reference to exemplary nucleic acid moleculesof the invention, the term is used in its usual biological sense, anddoes not refer to an entire multicellular organism, e.g., specificallydoes not refer to a human being. The cell can be present in an organism,e.g., birds, plants and mammals, such as humans, cows, sheep, apes,monkeys, swine, dogs, and cats. The cell can be prokaryotic (e.g.,bacterial cell) or eukaryotic (e.g., mammalian or plant cell). The cellcan be of somatic or germ line origin, totipotent or pluripotent,dividing or non-dividing. The cell can also be derived from or cancomprise a gamete or embryo, a stem cell, or a fully differentiatedcell.

The phrases “chemically-modified nucleotide,” “modified nucleotide” or,when used in reference to nucleotides of the invention, “chemicalmodification,” refer to a nucleotide that contains a modification in thechemical structure of the heterocyclic base moiety, sugar and/orphosphate of the unmodified (or natural) nucleotide as is generallyknown in the art (i.e., at least one modification compared to anaturally occurring RNA or DNA nucleotide). In certain embodiments, theterms can refer to certain forms of RNA that are naturally occurring incertain biological systems, for example 2′-O-methyl modifications orinosine modifications. A modified nucleotide includes abasicnucleotides. Modified nucleotides include nucleotides with a modifiedsugar ring or sugar surrogate. Modified heterocyclic base moietiesinclude without limitation, universal bases, hydrophobic bases,promiscuous bases, size-expanded bases, and fluorinated bases as definedherein. Certain of these nucleobases are particularly useful forincreasing the binding affinity of the siNA molecules as providedherein. These include 5-substituted pyrimidines, 6-azapyrimidines andN-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine,5-propynyluracil and 5-propynylcytosine. A modified internucleosidelinkage refers to any internucleoside linkage other than a naturallyoccurring internucleoside linkage. Non-limiting examples of modifiednucleotides are described herein and in U.S. application Ser. No.12/064,014 (published as US 20090176725).

The terms “complementarity” or “complementary” as used herein refers toits meaning as is generally accepted in the art. With reference toexemplary nucleic acid molecules of the invention, the terms generallyrefer to the formation or existence of hydrogen bond(s) between onenucleic acid sequence and another nucleic acid sequence by eithertraditional Watson-Crick or other non-traditional types of bonding asdescribed herein. In reference to the nucleic molecules of the presentinvention, the binding free energy for a nucleic acid molecule with itscomplementary sequence is sufficient to allow the relevant function ofthe nucleic acid to proceed, e.g., RNAi activity. Determination ofbinding free energies for nucleic acid molecules is well known in theart (see, e.g., Turner et al., 1987, CSH Symp. Quant. Biol. LII pp.123-133; Frier et al., 1986, Proc. Nat. Acad. Sci. USA 83:9373-9377;Turner et al., 1987, J. Am. Chem. Soc. 109:3783-3785). Perfectcomplementary means that all the contiguous residues of a nucleic acidsequence will hydrogen bond with the same number of contiguous residuesin a second nucleic acid sequence. Partial complementarity can includevarious mismatches or non-based paired nucleotides (e.g., 1, 2, 3, 4, 5,6, 7, 8, 9, 10 or more mismatches, non-nucleotide linkers, or non-basedpaired nucleotides) within the nucleic acid molecule, which can resultin bulges, loops, or overhangs between the sense strand or sense regionand the antisense strand or antisense region of a nucleic acid moleculeor between the antisense strand or antisense region of a nucleic acidmolecule and a corresponding target nucleic acid molecule. Such partialcomplementarity can be represented by a % complementarity that isdetermined by the number of non-base paired nucleotides, e.g., about50%, 60%, 70%, 80%, 90% etc. depending on the total number ofnucleotides involved. Such partial complementarity is permitted to theextent that the nucleic acid molecule (e.g., siNA) maintains itsfunction, for example the ability to mediate sequence specific RNAi.

The terms “composition” or “formulation” as used herein refer to theirgenerally accepted meaning in the art. These terms generally refer to acomposition or formulation, such as in a pharmaceutically acceptablecarrier or diluent, in a form suitable for administration, e.g.,systemic or local administration, into a cell or subject, including, forexample, a human. Suitable forms, in part, depend upon the use or theroute of entry, for example oral, transdermal, inhalation, or byinjection. Such forms should not prevent the composition or formulationfrom reaching a target cell (i.e., a cell to which the negativelycharged nucleic acid is desirable for delivery). For example,compositions injected into the blood stream should be soluble. Otherfactors are known in the art, and include considerations such astoxicity and forms that prevent the composition or formulation fromexerting its effect. As used herein, pharmaceutical formulations includeformulations for human and veterinary use. Non-limiting examples ofagents suitable for formulation with the nucleic acid molecules of theinstant invention include: lipid nanoparticles (see for example Sempleet al., 2010, Nat Biotechnol., 28(2):172-6); P-glycoprotein inhibitors(such as Pluronic P85); biodegradable polymers, such as poly(DL-lactide-coglycolide) microspheres for sustained release delivery(Emerich, D F et al., 1999, Cell Transplant, 8, 47-58); and loadednanoparticles, such as those made of polybutylcyanoacrylate. Othernon-limiting examples of delivery strategies for the nucleic acidmolecules of the instant invention include material described in Boadoet al., 1998, J. Pharm. Sci., 87, 1308-1315; Tyler et al., 1999, FEBSLett., 421, 280-284; Pardridge et al., 1995, PNAS USA., 92, 5592-5596;Boado, 1995, Adv. Drug Delivery Rev., 15, 73-107; Aldrian-Herrada etal., 1998, Nucleic Acids Res., 26, 4910-4916; and Tyler et al., 1999,PNAS USA., 96, 7053-7058. A “pharmaceutically acceptable composition” or“pharmaceutically acceptable formulation” can refer to a composition orformulation that allows for the effective distribution of the nucleicacid molecules of the instant invention to the physical location mostsuitable for their desired activity.

The term “conjugate” refers to an atom or group of atoms bound to ansiNA molecule of the invention. In general, conjugate groups modify oneor more properties of the molecule to which they are attached,including, but not limited to pharmacodynamics, pharmacokinetic,binding, absorption, cellular distribution, cellular uptake, charge andclearance. Conjugate groups are routinely used in the chemical arts andare linked directly or via an optional linking moiety or linking groupto the parent compound, such as an siNA molecule. In certainembodiments, conjugate groups includes without limitation,intercalators, reporter molecules, polyamines, polyamides, polyethyleneglycols, thioethers, polyethers, cholesterols, thiocholesterols, cholicacid moieties, folate, lipids, phospholipids, biotin, phenazine,phenanthridine, anthraquinone, adamantane, acridine, fluoresceins,rhodamines, coumarins and dyes. In certain embodiments, conjugates areattached to a 3′ or 5′ terminal nucleotide or to an internal nucleotidesof an siNA molecule. As used herein, “conjugate linking group” refers toany atom or group of atoms used to attach a conjugate to an siNAmolecule. Linking groups or bifunctional linking moieties such as thoseknown in the art are amenable to the present invention.

The term “cyano” refers to the group (—CN).

The terms “detecting” or “measuring,” as used herein in connection withan activity, response or effect, indicate that a test for detecting ormeasuring such activity, response, or effect is performed. Suchdetection and/or measuring may include values of zero. Thus, if a testfor detection or measuring results in a finding of no activity (activityof zero), the step of detecting or measuring the activity hasnevertheless been performed. For example, in certain embodiments, thepresent invention provides methods that comprise steps of detecting genesilencing activity. Any such step may include values of zero.

The term “expression” as used herein refers to its meaning as isgenerally accepted in the art. The term generally refers to the processby which a gene ultimately results in a protein. Expression includes,but is not limited to, transcription, splicing, post-transcriptionalmodification and translation.

The term “gene” as used herein refers to its meaning as is generallyaccepted in the art. The term generally refers to a nucleic acid (e.g.,DNA or RNA) sequence that comprises partial length or entire lengthcoding sequences necessary for the production of a polypeptide. A genecan also include the UTR or non-coding region of the nucleic acidsequence. A gene can also encode a functional RNA (fRNA) or non-codingRNA (ncRNA), such as small temporal RNA (stRNA), micro RNA (miRNA),small nuclear RNA (snRNA), short interfering RNA (siRNA), smallnucleolar RNA (snRNA), ribosomal RNA (rRNA), transfer RNA (tRNA) andprecursor RNAs thereof. Such non-coding RNAs can serve as target nucleicacid molecules for siNA mediated RNA interference in modulating theactivity of fRNA or ncRNA involved in functional or regulatory cellularprocesses. Aberrant fRNA or ncRNA activity leading to disease cantherefore be modulated by siNA molecules of the invention. siNAmolecules targeting fRNA and ncRNA can also be used to manipulate oralter the genotype or phenotype of a subject, organism or cell, byintervening in cellular processes such as genetic imprinting,transcription, translation, or nucleic acid processing (e.g.,transamination, methylation etc.). The term “gene” can be used whenreferencing a gene to which an siNA molecule of the invention is eitherdirectly (i.e., the siNA molecule comprises an antisense strand havingpartial or complete complementarity to the gene) or indirectly (i.e.,the siNA molecule comprises an antisense strand having partial orcomplete complementarity to a gene in the expression or activity pathwayof the gene) targeted.

As appreciated by those of skill in the art, “halo” or “halogen” as usedherein is intended to include chloro, fluoro, bromo and iodo.

The term “heteroaryl,” as used herein, represents a stable monocyclic orbicyclic ring of up to 7 atoms in each ring, wherein at least one ringis aromatic and contains from 1 to 4 heteroatoms selected from the groupconsisting of O, N and S. Heteroaryl groups within the scope of thisdefinition include but are not limited to: acridinyl, carbazolyl,cinnolinyl, quinoxalinyl, pyrrazolyl, indolyl, benzotriazolyl, furanyl,thienyl, benzothienyl, benzofuranyl, benzimidazolonyl, benzoxazolonyl,quinolinyl, isoquinolinyl, dihydroisoindolonyl, imidazopyridinyl,isoindolonyl, indazolyl, oxazolyl, oxadiazolyl, isoxazolyl, indolyl,pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl,tetrahydroquinoline. “Heteroaryl” is also understood to include theN-oxide derivative of any nitrogen-containing heteroaryl. In cases wherethe heteroaryl substituent is bicyclic and one ring is non-aromatic orcontains no heteroatoms, it is understood that attachment is via thearomatic ring or via the heteroatom containing ring, respectively.

The term “heterocycle” or “heterocyclyl,” as used herein, is intended tomean a 3- to 10-membered aromatic or nonaromatic heterocycle containingfrom 1 to 4 heteroatoms selected from the group consisting of O, N andS, and includes bicyclic groups. For the purposes of this invention, theterm “heterocyclic” is also considered to be synonymous with the terms“heterocycle” and “heterocyclyl” and is understood as also having thedefinitions set forth herein. “Heterocyclyl” therefore includes theabove mentioned heteroaryls, as well as dihydro and tetrahydro analogsthereof. Further examples of “heterocyclyl” include, but are not limitedto the following: azetidinyl, benzoimidazolyl, benzofuranyl,benzofurazanyl, benzopyrazolyl, benzotriazolyl, benzothiophenyl,benzoxazolyl, carbazolyl, carbolinyl, cinnolinyl, furanyl, imidazolyl,indolinyl, indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl,isoquinolyl, isothiazolyl, isoxazolyl, naphthpyridinyl, oxadiazolyl,oxooxazolidinyl, oxazolyl, oxazoline, oxopiperazinyl, oxopyrrolidinyl,oxomorpholinyl, isoxazoline, oxetanyl, pyranyl, pyrazinyl, pyrazolyl,pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl, pyrimidyl, pyrrolyl,quinazolinyl, quinolyl, quinoxalinyl, tetrahydropyranyl,tetrahydrofuranyl, tetrahydrothiopyranyl, tetrahydroisoquinolinyl,tetrazolyl, tetrazolopyridyl, thiadiazolyl, thiazolyl, thienyl,triazolyl, 1,4-dioxanyl, hexahydroazepinyl, piperazinyl, piperidinyl,pyridin-2-onyl, pyrrolidinyl, morpholinyl, thiomorpholinyl,dihydrobenzoimidazolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl,dihydrobenzoxazolyl, dihydrofuranyl, dihydroimidazolyl, dihydroindolyl,dihydroisooxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl,dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl,dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl,dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl,dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl,dioxidothiomorpholinyl, methylenedioxybenzoyl, tetrahydrofuranyl, andtetrahydrothienyl, and N-oxides thereof. Attachment of a heterocyclylsubstituent can occur via a carbon atom or via a heteroatom.

The term “hydroxyl” refers to the group (—OH).

The terms “including” (and any form thereof, such as “includes” and“include”), “comprising” (and any form thereof, such as “has” or “have”)or “containing” (and any form thereof (“contains” or “contain”) areinclusive and open-ended and do not exclude additional, unrecitedelements or method steps.

The terms “inhibit,” “down-regulate,” or “reduce” as used herein referto their meanings as generally accepted in the art. With reference tonucleic acid molecules of the invention, the term generally refers toreduction in the expression of a gene, or in the level of RNA moleculesor equivalent RNA molecules encoding one or more proteins or proteinsubunits, or in the activity of one or more proteins or proteinsubunits, below that observed in the absence of the nucleic acidmolecules (e.g., siNA) of the invention. Down-regulation can beassociated with post-transcriptional silencing, such as RNAi mediatedcleavage.

The terms “intermittent” or “intermittently” as used herein refer totheir meaning as generally accepted in the art. The terms generallyrefer to periodic stopping and starting at either regular or irregularintervals.

The terms “internucleoside linkage,” “internucleoside linker,”“internucleoside linking group,” “internucleotide linkage,”“internucleotide linker” or “internucleotide linking group” are usedherein interchangeably and refer to any linker or linkage between twonucleoside (i.e., a heterocyclic base moiety and a sugar moiety) units,as is known in the art, including, for example, but not as limitation,phosphate, analogs of phosphate, phosphonate, guanidium, hydroxylamine,hydroxylhydrazinyl, amide, carbamate, alkyl, and substituted alkyllinkages. Internucleoside linkages constitute the backbone of a nucleicacid molecule. In one aspect, a nucleotide of an siNA molecule of theinvention may be linked to a consecutive nucleotide through a linkagebetween the 3′-carbon of the sugar of the first nucleotide and the sugarmoiety of the second nucleotide (herein referred to as a 3′internucleoside linkage). A 3′-5′ internucleoside linkage, as usedherein, refers to an internucleoside linkage that links two consecutivenucleoside units, wherein the linkage is between the 3′ carbon of thesugar moiety of the first nucleoside and the 5′ carbon of the sugarmoiety of the second nucleoside. In another aspect, a nucleotide of ansiNA molecule of the invention may be linked to a consecutive nucleotidethrough a linkage between the 2′-carbon of the sugar of the firstnucleotide and the sugar moiety of the second nucleotide (hereinreferred to as a 2′ internucleoside linkage). A 2′-5′ internucleosidelinkage, as used herein, refers to an internucleoside linkage that linkstwo consecutive nucleoside units, wherein the linkage is between the 2′carbon of the sugar moiety of the first nucleoside and the 5′ carbon ofthe sugar moiety of the second nucleoside.

The terms “mammalian” or “mammal” as used herein refers to theirmeanings as generally accepted in the art. The terms generally refer toany warm blooded vertebrate species, such as a human, mouse, rat, dog,cat, hamster, guinea pig, rabbit, livestock, and the like.

The phrase “metered dose inhaler” or “MDI” refers to a unit comprising acan, a secured cap covering the can, and a formulation metering valvesituated in the cap. MDI systems include a suitable channeling device.Suitable channeling devices comprise for example, a valve actuator and acylindrical or cone-like passage through which medicament can bedelivered from the filled canister via the metering valve to the nose ormouth of a patient such as a mouthpiece actuator.

The term “microRNA” or “miRNA” as used herein refers to its meaning asis generally accepted in the art. The term generally refers to a smallnon-coding RNA that regulates the expression of target messenger RNAseither by mRNA cleavage, translational repression/inhibition orheterochromatic silencing (see for example Ambros, 2004, Nature, 431,350-355; Bartel, 2004, Cell, 116, 281-297; Cullen, 2004, Virus Research,102, 3-9; He et al., 2004, Nat. Rev. Genet., 5, 522-531; Ying et al.,2004, Gene, 342, 25-28; and Sethupathy et al., 2006, RNA, 12:192-197).The phenomenon of RNA interference includes the endogenously inducedgene silencing effects of miRNAs. As used herein, “microRNA mimetic”refers to an siNA molecule having a sequence that is at least partiallyidentical to that of a microRNA. In certain embodiments, a microRNAmimetic comprises the microRNA seed region of a microRNA. In certainembodiments, a microRNA mimetic modulates translation of more than onetarget nucleic acid.

The term “modulate” or “modulation” as used herein refers to its meaningas is generally accepted in the art. With reference to nucleic acidmolecules of the invention, the term refers to when the expression of agene, or the level of one or more RNA molecules (coding or non-coding),or the activity of one or more RNA molecules or proteins or proteinsubunits, is up-regulated or down-regulated, such that expression levelor activity is greater than or less than that observed in the absence ofthe molecule that effects modulation. For example, the term “modulate”in some embodiments can refer to inhibition and, in other embodiments,can refer to potentiation or up-regulation, e.g., of gene expression.

The phrase “non-base paired” refers to nucleotides that are not basepaired between the sense strand or sense region and the antisense strandor antisense region of a double-stranded siNA molecule. Non-base pairednucleotides can include, for example, but not as limitation, mismatches,overhangs, and single stranded loops.

The term “non-nucleotide” refers to any group or compound which can beincorporated into a polynucleotide chain in the place of one or morenucleotide units, such as for example but not limitation, abasicmoieties or alkyl chains. The group or compound is “abasic” in that itdoes not contain a commonly recognized nucleotide base, such asadenosine, guanine, cytosine, uracil or thymine and, therefore, lacks anucleobase at the 1′-position.

The term “nucleobase” is used herein to refer to the heterocyclic baseportion of a nucleotide. Nucleobases may be naturally occurring or maybe modified. In certain embodiments, a nucleobase may comprise any atomor group of atoms capable of hydrogen bonding to a base of anothernucleic acid.

The term “nucleotide” is used as is generally recognized in the art.Nucleotides generally comprise a heterocyclic base moiety (i.e., anucleobase), a sugar, and an internucleoside linkage, e.g., a phosphate.The base can be a natural base (standard), a modified base, or a baseanalog, as are well known in the art. Such bases are generally locatedat the 1′ position of a nucleotide sugar moiety. Additionally, thenucleotides can be unmodified or modified at the sugar, internucleosidelinkage, and/or base moiety, (also referred to interchangeably asnucleotide analogs, modified nucleotides, non-natural nucleotides,non-standard nucleotides and others; see, for example, U.S. applicationSer. No. 12/064,014 (published as US 20090176725)). A naturallyoccurring internucleoside linkage refers to a 3′ to 5′ phosphodiesterlinkage (also referred to herein as a 3′-5′ phosphodiester linkage).

The term “overhang” as used herein refers to its meaning as is generallyaccepted in the art. With reference to exemplary double stranded nucleicacid molecules, the term generally refers to the terminal portion of anucleotide sequence that is not base paired between the two strands of adouble-stranded nucleic acid molecule. Overhangs, when present, aretypically at the 3′-end of one or both strands in an siNA duplex.

The term “oxo” refers to the group (═O).

The term “parenteral” as used herein refers to its meaning as isgenerally accepted in the art. The term generally refers to methods ortechniques of administering a molecule, drug, agent, or compound in amanner other than through the digestive tract, and includesepicutaneous, subcutaneous, intravascular (e.g., intravenous),intramuscular, or intrathecal injection or infusion techniques and thelike.

The phrase “pharmaceutically acceptable carrier or diluent” as usedherein refers to its meaning as it generally accepted in the art. Thephrase generally refers to any substance suitable for use inadministering to an animal. In certain embodiments, a pharmaceuticallyacceptable carrier or diluent is sterile saline. In certain embodiments,such sterile saline is pharmaceutical grade saline.

The term “phosphorothioate” refers to an internucleoside phosphatelinkage comprising one or more sulfur atoms in place of an oxygen atom.Hence, the term phosphorothioate refers to both phosphorothioate andphosphorodithioate internucleoside linkages.

The term “position 1” refers to the position of the first nucleotide atthe 5′ end of an oligonucleotide strand, e.g., antisense strand. Allpositions referred to herein are the positions of a nucleotide countingfrom the 5′ end of an oligonucleotide strand, for example, positions 1-3of an antisense strand refer to the three nucleotides at positions 1, 2,and 3 counting from the 5′ end of the antisense strand. The term“5′-position 1 nucleotide” refers to the nucleotide at position 1 of anoligonucleotide strand that may or may not contain a 5′ cap. By way ofexample, a 5′ cap of a 5′-position 1 nucleotide can be anaturally-occurring 5′ phosphate cap or a modified terminal cap, asdefined herein. The term “position 21-3′ nucleotide” refers to thenucleotide at position 21 of an oligonucleotide strand that may or maynot further contain a 3′ cap.

The term “protecting group,” as used herein, refers to a labile chemicalmoiety that is known in the art to protect reactive groups, includingwithout limitation, hydroxyl, amino and thiol groups, against undesiredreactions during synthetic procedures. Protecting groups are typicallyused selectively and/or orthogonally to protect sites during reactionsat other reactive sites and can then be removed to leave the unprotectedgroup as is or available for further reactions. Protecting groups asknown in the art are described generally in Greene's Protective Groupsin Organic Synthesis, 4th edition, John Wiley & Sons, New York, 2007.Groups can be selectively incorporated into oligomeric compounds asprovided herein as precursors. For example, an amino group can be placedinto a compound as provided herein as an azido group that can bechemically converted to the amino group at a desired point in thesynthesis. Generally, groups are protected or present as precursors thatwill be inert to reactions that modify other areas of the parentmolecule for conversion into their final groups at an appropriate time.Further representative protecting or precursor groups are discussed inAgrawal et al, Protocols for Oligonucleotide Conjugates, Humana Press;New Jersey, 1994, 26, 1-72.

Examples of hydroxyl protecting groups include, without limitation,acetyl, t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl,1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, p-chlorophenyl,2,4-dinitrophenyl, benzyl, 2,6-dichlorobenzyl, diphenylmethyl,p-nitrobenzyl, bis(2-acetoxyethoxy)methyl (ACE), 2-trimethylsilylethyl,trimethylsilyl, triethylsilyl, t-butyldimethylsilyl,t-butyldiphenylsilyl, triphenylsilyl, [(triisopropylsilyl)oxy]methyl(TOM), benzoylformate, chloroacetyl, trichloroacetyl, trifluoroacetyl,pivaloyl, benzoyl, p-phenylbenzoyl, 9-fluorenylmethyl carbonate,mesylate, tosylate, triphenylmethyl (trityl), monomethoxytrityl,dimethoxytrityl (DMT), Irimethoxytrityl,1(2-fluorophenyl)-4-methoxypiperidin-4-yl (FPMP), 9-phenylxanthine-9-yl(Pixyl) and 9-(p-methoxyphenyl)xanthine-9-yl (MOX). Wherein morecommonly used hydroxyl protecting groups include, without limitation,benzyl, 2,6-dichlorobenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl,benzoyl, mesylate, tosylate, dimethoxytrityl (DMT),9-phenylxanthine-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthine-9-yl(MOX).

Examples of protecting groups commonly used to protect phosphate andphosphorus groups include without limitation, methyl, ethyl, benzyl(Bn), phenyl, isopropyl, fert-butyl, allyl, cyclohexyl (cHex),4-methoxybenzyl, 4-chlorobenzyl, 4-nitrobenzyl, 4-acyloxybenzyl,2-methylphenyl, 2,6-dimethylphenyl, 2-chlorophenyl, diphenylmethyl,4-methylthio-1-butyl, 2-(S-Acetylthio)ethyl (SATE), 2-cyanoethyl,2-cyano-1,1-dimethylethyl (CDM), 4-cyano-2-butenyl,2-(trimethylsilyl)ethyl (TSE), 2-(phenylthio)ethyl,2-(triphenylsilyl)ethyl, 2-(benzylsulfonyl)ethyl, 2,2,2-trichloroethyl,2,2,2-tribromoethyl, 2,3-dibromopropyl, 2,2,2-trifluoroethyl,thiophenyl, 2-chloro-4-tritylphenyl, 2-bromophenyl,2-[N-isopropyl-N-(4-methoxybenzoyl)amino]ethyl,4-(N-trifluoroacetylamino)butyl, 4-oxopentyl, 4-tritylaminophenyl,4-benzylaminophenyl and morpholino. Wherein more commonly used phosphateand phosphorus protecting groups include without limitation, methyl,ethyl, benzyl (Bn), phenyl, isopropyl, tert-butyl, 4-methoxybenzyl,4-chlorobenzyl, 2-chlorophenyl and 2-cyanoethyl.

Examples of amino protecting groups include, without limitation,carbamate-protecting groups, such as 2-trimethylsilylethoxycarbonyl(Teoc), 1-methyl-1-(4-biphenylyl)ethoxycarbonyl (Bpoc), t-butoxycarbonyl(BOC), allyloxycarbonyl (Alloc), 9-fluorenylmethyloxycarbonyl (Fmoc),and benzyl-oxycarbonyl (Cbz); amide-protecting groups, such as formyl,acetyl, trihaloacetyl, benzoyl, and nitrophenylacetyl;sulfonamide-protecting groups, such as 2-nitrobenzenesulfonyl; andimine- and cyclic imide-protecting groups, such as phthalimido anddithiasuccinoyl.

Examples of thiol protecting groups include without limitation,triphenylmethyl (trityl), benzyl (Bn), and the like. In certainembodiments, siNA molecules as provided herein can be prepared havingone or more optionally protected phosphorus containing internucleosidelinkages. Representative protecting groups for phosphorus containinginternucleoside linkages such as phosphodiester and phosphorothioatelinkages include β-cyanoethyl, diphenylsilylethyl, δ-cyanobutenyl, cyanop-xylyl (CPX), N-methyl-N-trifluoroacetyl ethyl (META), acetoxy phenoxyethyl (APE) and butene-4-yl groups. See for example U.S. Pat. Nos.4,725,677 and Re. 34,069 (β-cyanoethyl); Beaucage et al., Tetrahedron,1993, 49(10), 1925-1963; Beaucage et al, Tetrahedron, 1993, 49(46),10441-10488; Beaucage et al, Tetrahedron, 1992, 48(12), 2223-2311.

In certain embodiments, compounds having reactive phosphorus groups areprovided that are useful for forming internucleoside linkages includingfor example phosphodiester and phosphorothioate internucleosidelinkages. Such reactive phosphorus groups are known in the art andcontain phosphorus atoms in P^(III) or P^(V) valence state including,but not limited to, phosphoramidite, H-phosphonate, phosphate triestersand phosphorus containing chiral auxiliaries. In certain embodiments,reactive phosphorus groups are selected from diisopropylcyanoethoxyphosphoramidite (—O*—P[N[(CH(CH₃)₂]₂]O(CH₂)₂CN) and H-phosphonate(—O*—P(═O)(H)OH), wherein the O* is provided from the monomer. Apreferred synthetic solid phase synthesis utilizes phosphoramidites(P^(III) chemistry) as reactive phosphites. The intermediate phosphitecompounds are subsequently oxidized to the phosphate or thiophosphate(P^(V) chemistry) using known methods to yield phosphodiester orphosphorothioate internucleoside linkages. Additional reactivephosphates and phosphites are disclosed in Tetrahedron Report Number 309(Beaucage and Iyer, Tetrahedron, 1992, 48, 2223-2311).

The term “ribonucleotide” as used herein refers to its meaning as isgenerally accepted in the art. The term generally refers to a nucleotidewith a hydroxyl group at the 2′ position of a β-D-ribofuranose moiety.

The term “RNA” as used herein refers to its generally accepted meaningin the art. Generally, the term RNA refers to a molecule comprising atleast one ribofuranoside moiety. The term can include double-strandedRNA, single-stranded RNA, isolated RNA such as partially purified RNA,essentially pure RNA, synthetic RNA, recombinantly produced RNA, as wellas altered RNA that differs from naturally occurring RNA by theaddition, deletion, substitution and/or alteration of one or morenucleotides. Such alterations can include addition of non-nucleotidematerial, such as to the end(s) of an siNA molecule or internally, forexample at one or more nucleotides of the RNA. Nucleotides in thenucleic acid molecules of the instant invention can also comprisenon-standard nucleotides, such as non-naturally occurring nucleotides orchemically synthesized nucleotides or deoxynucleotides. These alteredRNAs can be referred to as analogs or analogs of naturally-occurringRNA.

The phrase “RNA interference” or term “RNAi” refer to the biologicalprocess generally known in the art of inhibiting or down regulating geneexpression in a cell, typically by causing destruction of specifictarget RNA and mediated by sequence-specific nucleic acid molecules(e.g., short interfering nucleic acid molecule), see for example Zamoreand Haley, 2005, Science, 309, 1519-1524; Vaughn and Martienssen, 2005,Science, 309, 1525-1526; Zamore et al., 2000, Cell, 101, 25-33; Bass,2001, Nature, 411, 428-429; Elbashir et al., 2001, Nature, 411, 494-498;and Kreutzer et al., International PCT Publication No. WO 00/44895;Zernicka-Goetz et al., International PCT Publication No. WO 01/36646;Fire, International PCT Publication No. WO 99/32619; Plaetinck et al.,International PCT Publication No. WO 00/01846; Mello and Fire,International PCT Publication No. WO 01/29058; Deschamps-Depaillette,International PCT Publication No. WO 99/07409; and Li et al.,International PCT Publication No. WO 00/44914; Allshire, 2002, Science,297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein,2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297,2232-2237; Hutvagner and Zamore, 2002, Science, 297, 2056-60; McManus etal., 2002, RNA, 8, 842-850; Reinhart et al., 2002, Gene & Dev., 16,1616-1626; and Reinhart & Bartel, 2002, Science, 297, 1831).Additionally, the term RNAi is meant to be equivalent to other termsused to describe sequence specific RNA interference, such as posttranscriptional gene silencing, translational inhibition,transcriptional inhibition, or epigenetics. For example, siNA moleculesof the invention can be used to epigenetically silence genes at eitherthe post-transcriptional level or the pre-transcriptional level. In anon-limiting example, epigenetic modulation of gene expression by siNAmolecules of the invention can result from siNA mediated modification ofchromatin structure or methylation patterns to alter gene expression(see, for example, Verdel et al., 2004, Science, 303, 672-676;Pal-Bhadra et al., 2004, Science, 303, 669-672; Allshire, 2002, Science,297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein,2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297,2232-2237). Modulation of gene expression by siNA molecules of theinvention can result from siNA mediated cleavage of RNA (either codingor non-coding RNA) via RISC.

The phrase “seed region” as used herein refers to its meaning as isgenerally accepted in the art. Generally, the phrase refers to a regionat or near the 5′ end of an antisense strand of an siNA molecule havinga nucleobase sequence that is important for target nucleic acidrecognition by the molecule. In certain embodiments, a seed regioncomprises nucleotides 2-8 (i.e., located from positions 2-8) of an siNAmolecule. In certain embodiments, a seed region comprises nucleotides2-7 of an siNA molecule. In certain embodiments, a seed region comprisesnucleotides 1-7 of an siNA molecule. In certain embodiments, a seedregion comprises nucleotides 1-6 of an siNA molecule. In certainembodiments, a seed region comprises nucleotides 1-8 of an siNAmolecule. As used herein, “microRNA seed region” refers to a seed regionof a microRNA or microRNA mimetic.

The phrase “sense region” as used herein refers to its meaning as isgenerally accepted in the art. With reference to nucleic acid moleculesof the invention, the term refers to a nucleotide sequence of an siNAmolecule having complementarity to an antisense region of the siNAmolecule. In addition, the sense region of a siNA molecule can comprisea nucleic acid sequence having homology or sequence identity with atarget nucleic acid sequence. In one embodiment, the sense region of thesiNA molecule is also referred to as the sense strand or passengerstrand.

The phrases “short interfering nucleic acid,” “siNA,” “siNA molecule,”“short interfering RNA,” “siRNA,” “short interfering nucleic acidmolecule,” “short interfering oligonucleotide molecule,” or “chemicallymodified short interfering nucleic acid molecule” refer to any nucleicacid molecule capable of inhibiting or down regulating gene expressionor viral replication by mediating RNA interference (“RNAi”) in asequence-specific manner. These terms can refer to both individualnucleic acid molecules, a plurality of such nucleic acid molecules, orpools of such nucleic acid molecules. The siNA can be a symmetric orasymmetric double-stranded nucleic acid molecule comprisingself-complementary sense and antisense strands or regions, wherein theantisense strand/region comprises a nucleotide sequence that iscomplementary to a nucleotide sequence in a target nucleic acid moleculeor a portion thereof, and the sense strand/region comprises a nucleotidesequence corresponding to the target nucleic acid sequence or a portionthereof. A symmetric duplex refers to an siNA molecule comprising senseand antisense regions each comprising the same number of nucleotides. Anasymmetric duplex refers to an siNA molecule comprising an antisenseregion and a sense region that comprises fewer nucleotides than theantisense region, to the extent that the sense region has enoughcomplementary nucleotides to base pair with the antisense region to forma duplex. For example, an asymmetric double-stranded siNA molecule ofthe invention can comprise an antisense region having length sufficientto mediate RNAi in a cell or in vitro system, e.g. about 15 to about 30,and a sense region having about 3 to about 25 nucleotides that arecomplementary to the antisense region. As an example, an asymmetricdouble-stranded hairpin siNA molecule can also comprise a loop regioncomprising about 4 to about 12 nucleotides. The loop portion of anasymmetric hairpin siNA molecule can comprise nucleotides,non-nucleotides, linker molecules, or conjugate molecules as describedherein. An siNA molecule of the invention can also comprise asingle-stranded polynucleotide having a nucleotide sequencecomplementary to a portion of a nucleotide sequence in a target nucleicacid molecule (for example, where such siNA molecule does not requirethe presence within the siNA molecule of a nucleotide sequencecorresponding to the target nucleic acid sequence or a portion thereof).A single-stranded siNA molecule is an RNAi molecule, functioning throughan RNAi mechanism.

The term “subject” as used herein refers to its meaning as is generallyaccepted in the art. The term generally refers to an organism to whichthe nucleic acid molecules of the invention can be administered. Asubject can be a mammal or mammalian cells, including human or humancells. The term also refers to an organism which is a donor or recipientof explanted cells or the cells themselves.

The term “sugar moiety” means a natural or modified sugar ring or sugarsurrogate.

The term “sugar surrogate” generally refers to a structure that iscapable of replacing the furanose ring of a naturally occurringnucleotide. In certain embodiments, sugar surrogates are non-furanose(or 4′-substituted furanose) rings or ring systems or open systems. Suchstructures include simple changes relative to the natural furanose ring,such as a 6-membered ring or may be more complicated as is the case withthe non-ring system used in peptide nucleic acid. Sugar surrogatesincludes without limitation morpholinos, cyclohexenyls andcyclohexitols. In most nucleotides having a sugar surrogate group, theheterocyclic base moiety is generally maintained to permithybridization.

The phrase “systemic administration” as used herein refers to itsmeaning as is generally accepted in the art. The term generally refersin vivo systemic absorption or accumulation of drugs in the blood streamfollowed by distribution throughout the entire body.

The term “target” cellular protein, peptide, or polypeptide, orpolynucleotide or nucleic acid (such as “target DNA,” “target RNA,”“target nucleic acid”), as used herein, refers to a protein or nucleicacid, respectively, of which siNA molecule of the invention may becapable of inhibiting or down regulating the expression. In certainembodiments, target RNA is mRNA, pre-mRNA, non-coding RNA, pri-microRNA,pre-microRNA, mature microRNA, promoter-directed RNA, or naturalantisense transcripts. A target gene can be a gene derived from a cell,an endogenous gene, a transgene, or exogenous genes such as genes of apathogen, for example a virus, which is present in the cell afterinfection thereof. The cell containing the target gene can be derivedfrom or contained in any organism, for example a plant, animal,protozoan, virus, bacterium, or fungus. Non-limiting examples of plantsinclude monocots, dicots, or gymnosperms. Non-limiting examples ofanimals include vertebrates or invertebrates. Non-limiting examples offungi include molds or yeasts. For a review, see for example Snyder andGerstein, 2003, Science, 300, 258-260. For example, a target nucleicacid can be a cellular gene (or mRNA transcribed from the gene) whoseexpression is associated with a particular disorder or disease state, ora nucleic acid molecule from an infectious agent. In certainembodiments, target nucleic acid is a viral or bacterial nucleic acid.As used herein, “target mRNA” refers to a pre-selected RNA molecule thatencodes a protein. As used herein, “target pre-mRNA” refers to apre-selected RNA transcript that has not been fully processed into mRNA.Notably, pre-RNA includes one or more intron. As used herein, “targetmicroRNA” refers to a pre-selected non-coding RNA molecule about 18-30nucleobases in length that modulates expression of one or more proteinsor to a precursor of such a non-coding molecule. As used herein, “targetpdRNA” refers to a pre-selected RNA molecule that interacts with one ormore promoter to modulate transcription. As used herein, “targetnon-coding RNA” refers to a pre-selected RNA molecule that is nottranslated to generate a protein. Certain non-coding RNA is involved inregulation of expression. The phrase “pathway target” refers to anytarget involved in pathways of gene expression or activity. For example,any given target can have related pathway targets that can includeupstream, downstream, or modifier genes in a biologic pathway. Thesepathway target genes can provide additive or synergistic effects in thetreatment of diseases, conditions, and traits herein.

The phrases “target site,” “target sequence” and “target nucleic acidsite” as used herein refer to their meanings as generally accepted inthe art. The term generally refers to a sequence within a target nucleicacid (e.g., RNA) that is “targeted,” e.g., for cleavage mediated by ansiNA molecule that contains sequences within its antisense region thatare complementary to the target sequence.

The phrase “therapeutically effective amount” as used herein refers toits meaning as is generally accepted in the art. The term generallyrefers to the amount of a molecule, compound or composition that willelicit the biological or medical response of a cell, tissue, system,animal or human that is be sought by the researcher, veterinarian,medical doctor or other clinician. For example, if a given clinicaltreatment is considered effective when there is at least a 25% reductionin a measurable parameter associated with a disease or disorder, atherapeutically effective amount of a drug for the treatment of thatdisease or disorder is that amount necessary to effect at least a 25%reduction in that parameter.

The phrase “universal base” as used herein refers to its meaning as isgenerally accepted in the art. The term universal base generally refersto nucleotide base analogs that form base pairs with each of the naturalDNA/RNA bases with little or no discrimination between them.Non-limiting examples of universal bases include C-phenyl, C-naphthyland other aromatic derivatives, inosine, azole carboxamides, andnitroazole derivatives such as 3-nitropyrrole, 4-nitroindole,5-nitroindole, and 6-nitroindole as known in the art (see, for example,Loakes, 2001, Nucleic Acids Research, 29, 2437-2447).

The term “up-regulate” as used herein refers to its meaning as isgenerally accepted in the art. With reference nucleic acid molecules ofthe invention, the term refers to an increase in either the expressionof a gene, or the level of RNA molecules or equivalent RNA moleculesencoding one or more proteins or protein subunits, or the activity ofone or more RNAs, proteins or protein subunits, above that observed inthe absence of the nucleic acid molecules (e.g., siNA) of the invention.In certain instances, up-regulation or promotion of gene expression withan siNA molecule is above that level observed in the presence of aninactive or attenuated molecule. In other instances, up-regulation orpromotion of gene expression with siNA molecules is above that levelobserved in the presence of, for example, an siNA molecule withscrambled sequence or with mismatches. In still other instances,up-regulation or promotion of gene expression with a nucleic acidmolecule of the instant invention is greater in the presence of thenucleic acid molecule than in its absence. In some instances,up-regulation or promotion of gene expression is associated withinhibition of RNA mediated gene silencing, such as RNAi mediatedcleavage or silencing of a coding or non-coding RNA target thatdown-regulates, inhibits, or silences the expression of the gene ofinterest to be up-regulated. The down-regulation of gene expression can,for example, be induced by a coding RNA or its encoded protein, such asthrough negative feedback or antagonistic effects. The down-regulationof gene expression can, for example, be induced by a non-coding RNAhaving regulatory control over a gene of interest, for example bysilencing expression of the gene via translational inhibition, chromatinstructure, methylation, RISC mediated RNA cleavage, or translationalinhibition. As such, inhibition or down-regulation of targets thatdown-regulate, suppress, or silence a gene of interest can be used toup-regulate expression of the gene of interest toward therapeutic use.

The term “vector” as used herein refers to its meaning as is generallyaccepted in the art. The term vector generally refers to any nucleicacid- and/or viral-based expression system or technique used to deliverone or more nucleic acid molecules.

B. siNA Molecules

The instant invention features single- or double-stranded siNA moleculescapable of mediating RNA interference comprising an antisense strandthat is complementary to a nucleic acid target and comprises a 5′modified nucleotide with a 2′ internucleoside linkage. The 5′ modifiednucleotide makes up position 1 at the 5′ end of the antisense strand ofthe siNA molecules of the invention (i.e., the first nucleotide of the5′ end of the strand). The siNA molecules of the invention can takedifferent oligonucleotide forms, including but not limited to shortinterfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA)and short hairpin RNA (shRNA) molecules.

In particular, short-interfering nucleic acid (siNA) molecules of theinvention comprise an antisense strand having a 5′ modified nucleotideat nucleotide position 1, wherein said 5′ modified nucleotide is linkedto a nucleotide at position 2 of the strand through a 2′ internucleosidelinkage and may contain a modified 5′ cap (i.e., other than a 5′phosphate cap). In one embodiment, the short interfering nucleic acid(siNA) molecules of the invention comprise an antisense strand having a5′ modified nucleotide having the structure of Formula II:

wherein:

A is —OC(R³)₂—, —C(R³)₂O—, —C(R³)₂—, —C(R³)₂C(R³)₂— or —CR³═CR³—;

B is any heterocyclic base moiety;

D¹ and D^(1′) are independently selected from hydroxyl, —OR⁴, —SR⁴, or—N(R⁴)₂;

E is O, S, —NR⁵, —N—N(R⁴)₂ or —N—OR⁴;

J is an internucleoside linking group linking the 5′ modified nucleotideof Formula II to the sugar moiety of an adjacent nucleotide of the siNAmolecule;

R¹ and R^(1′) are independently selected from H, hydroxyl, halogen, C₁₋₆alkyl, —OR⁶, —N(R⁶)₂, or together form ═O or ═CH₂;

R² is H, C₁₋₆ alkyl or C₂₋₆ alkenyl;

R³ and R⁵ are independently selected from H, hydroxyl, halogen, C₁₋₁₀alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, aryl,

wherein R′ is selected from H or C₁₋₄ alkyl (which is optionallysubstituted with one to three substituents independently selected from—SR¹⁰, aryl, heteroaryl, amino, hydroxyl, oxo or —NH—C═(NH)NH₂, whereinthe aryl and heteroaryl are optionally substituted with hydroxyl), andR″ is selected from H, C₁₋₁₈ alkyl or aryl;

R⁴ is independently selected from H, C₁₋₁₀ alkyl, C₂₋₆ alkenyl, C₂₋₆alkynyl, aryl,

wherein R′ is selected from H or C₁₋₄ alkyl (which is optionallysubstituted with one to three substituents independently selected from—SR¹¹, aryl, heteroaryl, amino, hydroxyl, oxo or —NH—C═(NH)NH₂, whereinthe aryl and heteroaryl are optionally substituted with hydroxyl), andR″ is selected from H, C₁₋₁₈ alkyl or aryl;

R⁶ is independently selected from H, C₁₋₆ alkyl (which is optionallysubstituted with —OR, —SR⁷, —N(R⁸)₂, or (═O)—NR⁹ or from one to threehalogen), C₂₋₆ alkenyl, C₂₋₆ alkynyl, aryl,

wherein R′ is selected from H or C₁₋₄ alkyl (which is optionallysubstituted with one to three substituents independently selected from—SR¹⁰, aryl, heteroaryl, amino, hydroxyl, oxo, —NH—C═(NH)NH₂, whereinthe aryl and heteroaryl are optionally substituted with hydroxyl), andR″ is selected from H, C₁₋₁₈ alkyl or aryl;

R⁷ is methyl, —CF₃, —N(R⁸)₂ or —CH₂—N(R⁸)₂;

R⁸ is independently selected from H or C₁₋₆ alkyl;

R⁹ is (R⁸)₂, —R⁸—(CH₂)₂—N(R⁸)₂ or —R⁸—C(═NR⁸)[N(R⁸)₂]; and,

R¹⁰ is H or C₁₋₄ alkyl.

In certain embodiments, the siNA molecules of the invention aresingle-stranded molecules. In other embodiments, the siNA molecules ofthe invention are double-stranded molecules, wherein saiddouble-stranded molecules comprises the antisense strand and a sensestrand, wherein the sense strand is partially or completelycomplementary to the antisense strand.

In certain embodiments of the siNA molecules, R³ of Formula II isindependently selected from H, hydroxy, F, Cl, C₁₋₆ alkyl, C₂₋₆ alkenyl,C₂₋₆ alkynyl, aryl,

wherein R′ is selected from H, C₁₋₄ alkyl,

and R″ is selected from H, C₁₋₄ alkyl or aryl.

In certain embodiments of the siNA molecules of the invention, A ofFormula II is —OCH₂—, —CH₂CH₂— or —CH═CH—. In further embodiments, if Ais —OCH₂—,

is not

In further embodiments, A is —CH═CH—. In further embodiments, A is—CH═CH—, E is O, and D¹ and D^(1′) are each hydroxyl.

In certain embodiments of the siNA molecules of the invention, B isuracil, thymine, cytosine, 5-methylcytosine, adenine or guanine. Infurther embodiments, B is thymine.

In certain embodiments of the siNA molecules of the invention, D¹ andD^(1′) are independently selected from hydroxyl, —OCH₃ or —OCH₂CH₃. Incertain embodiments, D¹ and D^(1′) are independently hydroxyl. Incertain embodiments, D¹ and D^(1′) are independently —OCH₃. In certainembodiments, D¹ and D^(1′) are independently —OCH₂CH₃.

In certain embodiments of the siNA molecules of the invention, E is O orS. In other embodiments, E is O.

In certain embodiments of the siNA molecules of the invention, J is aphosphodiester internucleoside linking group or a phosphorothioateinternucleoside linking group.

In certain embodiments of the siNA molecules of the invention, R¹ is Hor hydroxyl. In certain embodiments, R^(1′) is H, hydroxyl, halogen or—OR⁶. In certain embodiments, R^(1′) is halogen, —OCH₃, —OCH₂F, —OCHF₂,—OCF₃, —OCH₂CH₃, —O(CH₂)₂F, —OCH₂CHF₂, —OCH₂CF₃, —OCH₂—CH═CH₂,—O(CH₂)₂—OCH₃, —O(CH₂)₂—SCH₃, —O(CH₂)₂—OCF₃, —OCH₂C(═O)—N(H)CH₃ or—OCH₂C(═O)—N(H)—(CH₂)₂—N(CH₃)₂. In certain embodiments, R^(1′) is F or—OCH₃.

In certain embodiments, R² is H.

In certain embodiments, each of R¹, R^(1′) and R² is H.

In certain embodiments of the siNA molecules of the invention, B isthymine and R¹, R^(1′) and R² are each H.

In certain embodiments of the siNA molecules of the invention, the 5′modified nucleotide has is selected from:

wherein B is uracil, thymine, cytosine, 5-methylcytosine, adenine orguanine. In further embodiments, B is thymine.

In certain embodiments of the siNA molecules of the invention, the 5′modified nucleotide is

wherein B is uracil, thymine, cytosine, 5-methylcytosine, adenine orguanine. In further embodiments, B is thymine.

In certain embodiments of the siNA molecules of the invention, the 5′modified nucleotide is

wherein B is uracil, thymine, cytosine, 5-methylcytosine, adenine orguanine. In further embodiments, B is thymine.

In certain embodiments of the siNA molecules of the invention, the 5′modified nucleotide is

wherein B is uracil, thymine, cytosine, 5-methylcytosine, adenine orguanine. In further embodiments, B is thymine.

In certain embodiments of the siNA molecules of the invention, the 5′modified nucleotide is

wherein B is uracil, thymine, cytosine, 5-methylcytosine, adenine orguanine. In further embodiments, B is thymine.

The siNA molecules of the invention can be single-stranded ordouble-stranded nucleic acid molecules. The nucleic acid molecules ofthe invention may modulate expression of a nucleic acid target in a cellor animal. In one embodiment, the siNA molecules of the inventioninhibit or reduce expression of said nucleic acid target.

In one aspect, the invention provides single-stranded short interferingnucleic acid (siNA) molecules, wherein the single oligonucleotide strandcomprises a sequence that is complementary to at least a part of anucleic acid target sequence associated with gene expression. Forpurposes of this disclosure, the single strand of a single-stranded siNAmolecule of the invention is referred to as the antisense strand.

In another aspect, the invention provides double-stranded shortinterfering nucleic acid (siNA) molecules, wherein a double-strandedsiNA molecule comprises a sense and an antisense oligonucleotide strand.The antisense strand comprises a sequence that is complementary to atleast a part of a nucleic acid target associated with gene expression,and the sense strand is complementary to the antisense strand. Thedouble-stranded siNA molecules of the invention can comprise twodistinct and separate strands that can be symmetric or asymmetric andare complementary, i.e., two single-stranded oligonucleotides, or cancomprise one single-stranded oligonucleotide in which two complementaryportions, e.g., a sense region and an antisense region (which, in thiscontext, will be referred to herein as a sense strand and an antisensestrand, respectively), are base-paired, and are covalently linked by oneor more single-stranded “hairpin” areas (i.e. loops) resulting in, forexample, a short-hairpin polynucleotide.

The linker can be polynucleotide linker or a non-nucleotide linker. Insome embodiments, the linker is a non-nucleotide linker. In someembodiments, a hairpin siNA molecule of the invention contains one ormore loop motifs, wherein at least one of the loop portions of the siNAmolecule is biodegradable. For example, a short hairpin siNA molecule ofthe invention is designed such that degradation of the loop portion ofthe siNA molecule in vivo can generate a double-stranded siNA moleculewith 3′-terminal overhangs, such as 3′-terminal nucleotide overhangscomprising 1, 2, 3 or 4 nucleotides.

The antisense strand of the siNA molecules of the invention iscomplementary to a portion of a target nucleic acid sequence. In someembodiments, the target nucleic acid is selected from a target mRNA, atarget pre-mRNA, a target microRNA, and a target non-coding RNA. Incertain embodiments, the antisense strand of the siNA molecules of theinvention comprises a region that is 100% complementarity to a targetnucleic acid sequence and wherein the region of 100% complementarity isat least 10 nucleobases. In certain embodiments, the region of 100%complementarity is at least 15 nucleobases. In certain embodiments, theregion of 100% complementarity is at least 20 nucleobases. In certainembodiments, the region of 100% complementarity is at least 25nucleobases. In certain embodiments, the region of 100% complementarityis at least 30 nucleobases. In certain embodiments, the antisense strandof the siNA molecules of the invention is at least 85% complementary toa target nucleic acid sequence. In certain embodiments, the antisensestrand is at least 90% complementary to a target nucleic acid sequence.In certain embodiments, the antisense strand is at least 95%complementary to a target nucleic acid sequence. In certain embodiments,the antisense strand is at least 98% complementary to a target nucleicacid sequence. In certain embodiments, the antisense strand is 100%complementary to a target nucleic acid sequence. The complementarynucleotides may or may not be contiguous nucleotides. In one embodiment,the complementary nucleotides are contiguous nucleotides.

In certain embodiments, the siNA molecules of the invention have betweenabout 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, or 30) nucleotides in the antisense strand thatare complementary to a nucleotide sequence of a target nucleic acidmolecule. In certain embodiments, the siNA molecules of the inventioncomprise an antisense strand having at least 15 nucleotides havingsequence complementarity to a target sequence. In certain embodiments,the siNA molecules of the invention comprise an antisense strand havingat least 18 nucleotides having sequence complementarity to a targetsequence. In certain embodiments, the siNA molecules of the inventioncomprise an antisense strand having at least 19 nucleotides havingsequence complementarity to a target sequence. In certain embodiments,the siNA molecules of the invention comprise an antisense strand havingat least 20 nucleotides having sequence complementarity to a targetsequence. In certain embodiments, the siNA molecules of the inventioncomprise an antisense strand having at least 21 nucleotides havingsequence complementarity to a target sequence. In certain embodiments ofthis aspect of the invention, the complementary nucleotides arecontiguous nucleotides.

In some embodiments, double-stranded siNA molecules of the inventionhave perfect complementarity between the sense strand or sense regionand the antisense strand or antisense region of the siNA molecule, withthe exception of any overhanging region.

In yet other embodiments, double-stranded siNA molecules of theinvention have partial complementarity (i.e., less than 100%complementarity) between the sense strand or sense region and theantisense strand or antisense region of the siNA molecule. Thus, in someembodiments, the double-stranded nucleic acid molecules of theinvention, have between about 15 to about 30 (e.g., about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides inone strand (e.g., sense strand) that are complementary to thenucleotides of the other strand (e.g., antisense strand). In certainembodiments, the double-stranded siNA molecules of the invention have 17nucleotides in the sense region that are complementary to nucleotides ofthe antisense region of the nucleic acid molecule. In certainembodiments, the double-stranded siNA molecules of the invention have 18nucleotides in the sense region that are complementary to nucleotides ofthe antisense region of the nucleic acid molecule. In certainembodiments, the double-stranded siNA molecules of the invention have 19nucleotides in the sense region that are complementary to nucleotides ofthe antisense region of the nucleic acid molecule. In certainembodiments, the double-stranded siNA molecules of the invention have 20nucleotides in the sense region that are complementary to nucleotides ofthe antisense region of the nucleic acid molecule. In certainembodiments of this aspect of the invention, the complementarynucleotides between the strands are contiguous nucleotides.

In symmetric siNA molecules of the invention, each strand, the sense(passenger) strand and antisense (guide) strand, are independently about15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30) nucleotides in length. Generally, each strand ofa symmetric siNA molecule of the invention is about 19-24 (e.g., about19, 20, 21, 22, 23 or 24) nucleotides in length. In certain embodiments,each strand of a symmetric siNA molecule of the invention is 19nucleotides in length. In certain embodiments, each strand of asymmetric siNA molecule of the invention is 20 nucleotides in length. Incertain embodiments, each strand of a symmetric siNA molecule of theinvention is 21 nucleotides in length. In certain embodiments, eachstrand of a symmetric siNA molecule of the invention is 22 nucleotidesin length.

In asymmetric siNA molecules of the invention, the antisense strand ofthe molecule is about 15 to about 30 (e.g., about 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in length,wherein the sense region is about 3 to about 25 (e.g., about 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or25) nucleotides in length. Generally, the antisense strand of anasymmetric siNA molecules of the invention is about 19-24 (e.g., about19, 20, 21, 22, 23 or 24) nucleotides in length. In one embodiment, thesense strand of an asymmetric siNA molecule of the invention can beabout 19-24 (e.g., about 19, 20, 21, 22, 23 or 24) nucleotides inlength.

In yet other embodiments, siNA molecules of the invention comprisehairpin siNA molecules, wherein the siNA molecules are about 25 to about70 (e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 40, 45,50, 55, 60, 65, or 70) nucleotides in length.

In certain embodiments, an siNA molecule of the invention is a microRNAmimetic, having a nucleotide sequence comprising a nucleotide portionthat is fully or partially identical to a seed region of a microRNA. Incertain embodiments, the nucleotide sequence of a microRNA mimetic has anucleotide portion that is 100% identical to a seed region of amicroRNA. In certain embodiments, the nucleotide sequence of a microRNAmimetic has a nucleotide portion that is at least 75% identical (e.g.,about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) to a seed region ofa microRNA. In certain embodiments, the nucleotide sequence of amicroRNA mimetic has a nucleotide portion that is 75% identical to aseed region of a microRNA. In certain embodiments, the nucleotidesequence of a microRNA mimetic has a nucleotide portion that is 80%identical to a seed region of a microRNA. In certain embodiments, thenucleotide sequence of a microRNA mimetic has a nucleotide portion thatis 90% identical to a seed region of a microRNA. In certain embodiments,the nucleotide sequence of a microRNA mimetic has a nucleotide portionthat is 95% identical to a seed region of a microRNA.

In other embodiments, siNA molecules of the invention can contain one ormore nucleotide deletions, substitutions, mismatches and/or additions(in reference to a target site sequence, or between strands of a duplexsiNA molecule); provided, however, that the siNA molecule maintains itsactivity, for example, to mediate RNAi. In a non-limiting example, thedeletion, substitution, mismatch and/or addition can result in a loop orbulge, or alternately a wobble or other alternative (non Watson-Crick)base pair. Thus, in some embodiments, for example, the double-strandednucleic acid molecules of the invention, have one or more (e.g., 1, 2,3, 4, 5, or 6) nucleotides in one strand or region (e.g., sense strand)that are mismatches or non-base-paired with the other strand or region(e.g., antisense strand). In certain embodiments, the siNA molecules ofthe invention contain no more than 3 mismatches. If the antisense strandof an siNA molecule contains mismatches to a target sequence, it ispreferable that the area of mismatch is not located in the center of acontiguous region of complementarity.

In certain embodiments, the siNA molecules of the invention compriseoverhangs of about 1 to about 4 (e.g., about 1, 2, 3 or 4) nucleotides.The nucleotides in the overhangs can be the same or differentnucleotides. In some embodiments, the overhangs occur at the 3′-end (orthe 3′ terminus) of one or both strands of double-stranded nucleic acidmolecules of the invention. For example, a double-stranded nucleic acidmolecule of the invention can comprise a nucleotide or non-nucleotideoverhang at the 3′-end of the antisense strand/region, at the 3′-end ofthe sense strand/region, or at the 3′ ends of both the antisensestrand/region and the sense strand/region. Overhanging nucleotides canbe modified or unmodified.

In some embodiments, the nucleotides comprising the overhanging portionof an siNA molecule of the invention comprise sequences based on antarget nucleic acid sequence in which the nucleotides comprising theoverhanging portion of the antisense strand/region are complementary tonucleotides in the target polynucleotide sequence and/or the nucleotidescomprising the overhanging portion of the sense strand/region comprisenucleotides from the target polynucleotide sequence. Thus, in someembodiments, the overhang comprises a two nucleotide overhang that iscomplementary to a portion of the target polynucleotide sequence. Inother embodiments, however, the overhang comprises a two nucleotideoverhang that is not complementary to a portion of the target nucleicacid sequence. In certain embodiments, the overhang comprises a 3′-UUoverhang that is not complementary to a portion of the target nucleicacid sequence. In other embodiments, the overhang comprises a UUoverhang at the 3′ end of the antisense strand and a TT overhang at the3′ end of the sense strand.

In any of the embodiments of the siNA molecules described herein having3′-end nucleotide overhangs, the overhangs are optionally chemicallymodified at one or more nucleic acid sugar, base, or backbone positions.Representative, but not limiting examples of modified nucleotides in theoverhanging portion of a double-stranded siNA molecule of the inventioninclude: 2′-O-alkyl (e.g., 2′-O-methyl), 2′-deoxy, 2′-deoxy-2′-fluoro,2′-deoxy-2′-fluoroarabino (FANA), 4′-thio, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, universalbase, acyclic, or 5-C-methyl nucleotides. In more preferred embodiments,the overhang nucleotides are each independently, a 2′-O-alkylnucleotide, a 2′-O-methyl nucleotide, a 2′-deoxy-2-fluoro nucleotide, ora 2′-deoxy ribonucleotide. In some instances the overhanging nucleotidesare linked by one or more phosphorothioate linkages.

In yet other embodiments, siNA molecules of the invention compriseduplex nucleic acid molecules with blunt ends (i.e., without nucleotideoverhangs), where both termini of the molecule are blunt, oralternatively, where one of the ends is blunt. In some embodiments, thesiNA molecules of the invention comprise one blunt end, for examplewherein the 5′-end of the antisense strand and the 3′-end of the sensestrand do not have any overhanging nucleotides, or wherein the 3′-end ofthe antisense strand and the 5′-end of the sense strand do not have anyoverhanging nucleotides. In other embodiments, siNA molecules of theinvention comprise two blunt ends, for example wherein the 3′-end of theantisense strand and the 5′-end of the sense strand, as well as the5′-end of the antisense strand and 3′-end of the sense strand, do nothave any overhanging nucleotides.

In any of the embodiments or aspects of the siNA molecules of theinvention, the sense strand and/or the antisense strand can further havea cap, such as described herein or as known in the art. A cap can bepresent at the 3′-end of the antisense strand, the 5′-end of the sensestrand, and/or the 3′-end of the sense strand. In the case of a hairpinsiNA molecule, a cap can be present at the 3′-end of the polynucleotide.The cap at the 5′-end of the antisense strand of an siNA of theinvention is encompassed by the 5′ modified nucleotide as set forth bythe structure of Formula I and II. In some embodiments, a cap is at oneor both ends of the sense strand of a double-stranded siNA molecule. Inother embodiments, a cap is at the 3′-end of antisense (guide) strand.In other embodiments, a cap is at the 3′-end of the sense strand and atthe 5′-end of the sense strand. Representative but non-limiting examplesof such terminal caps include an inverted abasic nucleotide andderivatives thereof (e.g., an inverted deoxy abasic nucleotide, atetra-N-acethylgalactosamine aminohexyl phosphate inverted abasicnucleotide (e.g., tetraGalNAcLys-6amiL-iB-omeC; see Table 14, infra), aninverted nucleotide moiety, a glyceryl modification, an alkyl orcycloalkyl group, a heterocycle or any other cap as is generally knownin the art.

Any of the embodiments of the siNA molecules of the invention can have a5′ phosphate terminus. In some embodiments, the siNA molecules lackterminal phosphates.

In certain embodiments, the double-stranded siNA molecules of theinvention comprise about 3 to about 30 (e.g., about 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, or 30) base pairs. Generally, the duplex structure of siNAs ofthe invention is between 15 and 30 base pairs, more generally between 18and 25 base pairs, yet more generally between 19 and 24 base pairs, andmost generally between 19 and 21 base pairs in length. In oneembodiment, a double-stranded siNA molecule of the invention comprises19 base pairs. In one embodiment, a double-stranded siNA molecule of theinvention comprises 20 base pairs. In one embodiment, a double-strandedsiNA molecule of the invention comprises 21 base pairs. Thedouble-stranded siNA molecules of this portion of the invention can beasymmetric or symmetric. In other embodiments of this aspect of theinvention, the siNA duplex molecules are hairpin structures.

Any siNA molecule of the invention can comprise one or morechemically-modified nucleotides in addition to the 5′ modifiednucleotide of the antisense strand, as described in detail supra.Modifications can be used to improve in vitro or in vivo characteristicssuch as stability, activity, toxicity, immune response (e.g., preventstimulation of an interferon response, an inflammatory orpro-inflammatory cytokine response, or a Toll-like Receptor response),and/or bioavailability. Various chemically modified siNA motifsdisclosed herein have the potential to maintain an RNAi activity that issubstantially similar to either unmodified or minimally-modified activesiRNA (see for example Elbashir et al., 2001, EMBO J., 20:6877-6888)while, at the same time, providing nuclease resistance andpharmacokinetic properties suitable for use in therapeutic applications.

In certain embodiments of the siNA molecules of the invention, whereinsuch siNA molecules comprise a 5′ modified nucleotide having thestructure of Formula II, any (e.g., one, more or all) additionalnucleotides present in the antisense and/or sense strand may be modifiednucleotides (e.g., wherein one additional nucleotide is modified, someadditional nucleotides (i.e., a plurality or more than one) aremodified, or all nucleotides of the molecule are modified nucleotides).Modifications include sugar modifications, base modifications, backbone(internucleoside linkage) modifications, non-nucleotide modifications,and/or any combination thereof. In certain embodiments, the siNAmolecules of the invention further comprise one or more additional 2′internucleoside linkages (e.g., one or more additional 2′-5′internucleoside linkages).

Non-limiting examples of chemical modifications that are suitable foruse in the siNA molecules of the invention are disclosed in U.S. Pat.No. 8,202,979 and U.S. patent application Ser. Nos. 10/981,966 and12/064,014 (published as US 20050266422 and US 20090176725,respectively), and in references cited therein, and include sugar, base,and backbone modifications, non-nucleotide modifications, and/or anycombination thereof. These U.S. Patents and Applications areincorporated hereby as references for the purpose of describing chemicalmodifications that are suitable for use with the siNA molecules of theinvention.

The chemical modifications of nucleotides present within a single siNAmolecule can be the same or different. In some embodiments, at least onestrand of an siNA molecule of the invention has at least one chemicalmodification. In other embodiments, each strand has at least onechemical modification, which can be the same or different, such assugar, base, or backbone (i.e., internucleotide linkage) modifications.In other embodiments, siNA molecules of the invention contain at least2, 3, 4, 5, or more different chemical modifications.

In some embodiments, the siNA molecules of the invention are partiallymodified (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55,or 59 nucleotides are modified) with chemical modifications. In someembodiments, an siNA molecule of the invention comprises at least about8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42,44, 46, 48, 50, 52, 54, 56, 58, or 60 nucleotides that are modifiednucleotides, excluding the 5′ modified nucleotide of the antisensestrand. In other embodiments, the siNA molecules of the invention arecompletely modified (100% modified) with chemical modifications, i.e.,the siNA molecule does not contain any ribonucleotides. In some ofembodiments, one or more of the nucleotides in the sense strand of thesiNA molecules of the invention are modified. In the same or otherembodiments, one or more of the nucleotides in the antisense strand ofthe siNA molecules of the invention are modified, excluding the 5′modified nucleotide of the antisense strand. In some embodiments, one ormore (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) of the nucleotidepositions independently in either one or both strands of an siNAmolecule of the invention are modified, excluding the 5′ modifiednucleotide of the antisense strand.

Modified nucleotides contained within the siNA molecules of the presentinvention include those with modifications at the 2′-carbon of a sugarmoiety and/or the 3′-carbon of a sugar moiety of a nucleotide. Incertain specific embodiments of the invention, at least one modifiednucleotide is a 2′-deoxy-2-fluoro nucleotide, a 2′-deoxy nucleotide, a2′-O-alkyl (e.g., 2′-O-methyl) nucleotide, a 2′-methoxyethoxy or alocked nucleic acid (LNA) nucleotide, as is generally recognized in theart.

In yet other embodiment of the invention, at least one nucleotide has aribo-like, Northern or A form helix configuration (see e.g., Saenger,Principles of Nucleic Acid Structure, Springer-Verlag ed., 1984).Non-limiting examples of nucleotides having a Northern configurationinclude locked nucleic acid (LNA) nucleotides (e.g., 2′-O,4′-C-methylene-(D-ribofuranosyl) nucleotides); 2′-methoxyethoxy (MOE)nucleotides; 2′-methyl-thio-ethyl nucleotides; 2′-deoxy-2′-fluoronucleotides; 2′-deoxy-2′-chloro nucleotides; 2′-azido nucleotides;2′-O-trifluoromethyl nucleotides; 2′-O-ethyl-trifluoromethoxynucleotides; 2′-O-difluoromethoxy-ethoxy nucleotides; 4′-thionucleotides; and 2′-O-methyl nucleotides. In various embodiments, amajority (e.g., greater than 50%) of the pyrimidine nucleotides presentin a double-stranded siNA molecule comprises a sugar modification. Insome of the same and/or other embodiments, a majority (e.g., greaterthan 50%) of the purine nucleotides present in a double-stranded siNAmolecule comprises a sugar modification.

In certain instances, purine and pyrimidine nucleotides of the siNAmolecules of the invention are differentially modified. In one example,purine and pyrimidine nucleotides can be differentially modified at the2′-carbon of the sugar moiety (i.e., at least one purine has a differentmodification from at least one pyrimidine in the same or differentstrand at the 2′-carbon of the sugar moiety). In certain embodiments,the purines are unmodified in one or both strands, while the pyrimidinesin one or both strands are modified. In certain other instances, thepyrimidines are unmodified in one or both strands, while the purines inone or both strands are modified. In certain instances, wherein the siNAmolecules comprise one or more modifications as described herein, thenucleotides at positions 2 and 3 at the 5′ end of the antisense (guide)strand are unmodified.

In some embodiments of the siNA molecules of the invention, thepyrimidine nucleotides in the antisense strand are 2′-O-methyl or2′-deoxy-2′-fluoro pyrimidine nucleotides, and the purine nucleotidespresent in the antisense strand are 2′-O-methyl nucleotides or 2′-deoxynucleotides. In certain embodiments, all of the pyrimidine nucleotidesin a complementary region of an antisense strand of an siNA molecule ofthe invention are 2′-deoxy-2′-fluoro pyrimidine nucleotides. In certainembodiments, all of the purines in the complementary region on theantisense strand are 2′-O-methyl purine nucleotides.

In other embodiments of the siNA molecules of the invention, thepyrimidine nucleotides in the sense strand are 2′-deoxy-2′-fluoropyrimidine nucleotides, and the purine nucleotides present in the sensestrand are 2′-O-methyl or 2′-deoxy purine nucleotides. In certainembodiments of the invention, all the pyrimidine nucleotides in thecomplementary region on the sense strand are 2′-deoxy-2′-fluoropyrimidine nucleotides. In certain embodiments, all the purinenucleotides in the complementary region on the sense strand are 2′-deoxypurine nucleotides.

In certain embodiments, all of the pyrimidine nucleotides in thecomplementary regions on the sense strand are 2′-deoxy-2′-fluoropyrimidine nucleotides; all of the pyrimidine nucleotides in thecomplementary region of the antisense strand are 2′-deoxy-2′-fluoropyrimidine nucleotides; all the purine nucleotides in the complementaryregion on the sense strand are 2′-deoxy purine nucleotides and all ofthe purines in the complementary region on the antisense strand are2′-O-methyl purine nucleotides.

In some embodiments, at least 5 or more of the pyrimidine nucleotides inone or both strands of an siNA molecule of the invention are2′-deoxy-2′-fluoro pyrimidine nucleotides. In some embodiments, at least5 or more of the pyrimidine nucleotides in one or both strands are2′-O-methyl pyrimidine nucleotides. In some embodiments, at least 5 ormore of the purine nucleotides in one or both strands are2′-deoxy-2′-fluoro purine nucleotides In some embodiments, at least 5 ormore of the purine nucleotides in one or both strands are 2′-O-methylpurine nucleotides.

In certain embodiments, the siNA molecules of the invention comprise oneor more modified internucleoside linking group (i.e., other than the 2′internucleoside linkage as set forth in the 5′ modified nucleotide ofFormula II). A modified internucleoside linking group is a linking groupother than a phosphodiester 3′-5′ internucleoside linking group,including but not limited to 2′ internucleoside linking groups (e.g.,phosphodiester and phosphorothioate 2′-5′ internucleoside linkages). Incertain embodiments, each internucleoside linking group is,independently, a 2′ or 3′ phosphodiester or phosphorothioateinternucleoside linking group. In certain embodiments, the 5′-mostinternucleoside linking group on either or both strands of an siNAmolecule of the invention is a phosphorothioate linking group. Incertain embodiments, the siNA molecules of the invention comprise from 3to 12 contiguous phosphorothioate linking groups, wherein thephosphorothioate linking groups are either 2′ or 3′ internucleosidelinking groups. In certain embodiments, the siNA molecules of theinvention comprise from 6 to 8 contiguous phosphorothioate linkinggroups, wherein the phosphorothioate linking groups are either 2′ or 3′internucleoside linking groups. In certain embodiments, the 3′ end ofthe antisense and/or sense strand of the siNA molecules of the inventioncomprises a phosphorothioate linking groups. In certain embodiments, thesiNA molecules of the invention comprise from 6 to 8 contiguousphosphorothioate linking groups at the 3′ end of the antisense and/orsense strand, wherein the phosphorothioate linking groups are either 2′or 3′ internucleoside linking groups.

In certain embodiments, an siNA molecule of the invention does notcontain any additional chemically-modified nucleotides with a 2′-5′internucleoside linkage (i.e., only the 5′ modified nucleotide atposition 1 of the antisense strand of an siNA molecule of the inventionhas a 2′ internucleoside linkage, as described by the 5′ modifiednucleotide of Formula II). In further embodiments, any one or moreadditional chemically-modified nucleotides in the antisense strandand/or, optionally, the sense strand of either a single- ordouble-stranded siNA molecule of the invention does not have a 2′-5′internucleoside linkage. In one embodiment of the siNA molecules of theinvention, the nucleotide at position two of the antisense strand doesnot contain a 2′-5′ internucleoside linkage. In another embodiment, thenucleotide at position three of the antisense strand does not contain a2′-5′ internucleoside linkage. In a further embodiment, neithernucleotide at position two nor position 3 contain a 2′-5′internucleoside linkage.

In one embodiment, the siNA molecules of the invention, comprising anantisense strand having a 5′ modified nucleotide of the structure ofFormula II, comprise one or more additional nucleotides that are linkedto an adjacent (consecutive) nucleotide through a 2′ internucleosidelinkage. In particular, in certain embodiments, the siNA molecules ofthe invention further comprise one or more additional nucleotides havingthe structure of Formula III:

wherein:

B_(x) is any heterocyclic base moiety;

J_(x) is an internucleoside linking group linking the nucleotide ofFormula III to the sugar moiety of the adjacent (consecutive) nucleotideof the siNA molecule;

R_(x) ¹ and R_(x) ^(1′) are independently selected from H, hydroxyl,halogen, C₁₋₆ alkyl, —OR_(x) ³, —N(R_(x) ³)₂, or together form ═O or═CH₂;

R_(x) ² is H, C₁₋₆ alkyl or C₂₋₆ alkenyl;

R_(x) ³ is independently selected from H, C₁₋₆ alkyl (which isoptionally substituted with —OR_(x) ⁴, —SR_(x) ⁴, —N(R_(x) ⁵)₂,(═O)—NR_(x) ⁶ or from one to three halogen), C₂₋₆ alkenyl, C₂₋₆ alkynyl,aryl,

wherein R_(x)′ is selected from H or C₁₋₄ alkyl (which is optionallysubstituted with one to three substituents independently selectedfrom—SR_(x) ⁷, aryl, heteroaryl, amino, hydroxyl, oxo, —NH—C═(NH)NH₂,wherein the aryl and heteroaryl are optionally substituted withhydroxyl), and R_(x)″ is selected from H, C₁₋₁₈ alkyl or aryl;

R_(x) ⁴ is methyl, —CF₃, —N(R_(x) ⁵)₂ or —CH₂—N(R_(x) ⁵)₂;

R_(x) ⁵ is independently selected from H or C₁₋₆ alkyl;

R_(x) ⁶ is (R_(x) ⁵)₂, —R_(x) ⁵—(CH₂)₂—N(R_(x) ⁵)₂ or —R_(x) ⁵—C(═NR_(x)⁵)[N(R_(x) ⁵)₂]; and,

R_(x) ⁷ is H or C₁₋₄ alkyl.

In certain embodiments of this aspect of the invention, B_(x) is uracil,thymine, cytosine, 5-methylcytosine, adenine or guanine.

In certain embodiments of this aspect of the invention, J_(x) is aphosphodiester internucleoside linking group or a phosphorothioateinternucleoside linking group.

In certain embodiments of this aspect of the invention, R_(x) ¹ is H. Incertain embodiments of this aspect of the invention, R_(x) ^(1′) is H,halogen or —OR_(x) ³. In other embodiments of this aspect of theinvention, R_(x) ^(1′) is halogen, —OCH₃, —OCH₂F, —OCHF₂, —OCF₃,—OCH₂CH₃, —O(CH₂)₂F, —OCH₂CHF₂, —OCH₂CF₃, —OCH₂—CH═CH₂, —O(CH₂)₂—OCH₃,—O(CH₂)₂—SCH₃, —O(CH₂)₂—OCF₃, —OCH₂C(═O)—N(H)CH₃ or—OCH₂C(═O)—N(H)—(CH₂)₂—N(CH₃)₂. In other embodiments of this aspect ofthe invention, R_(x) ^(1′) is H, F, hydroxyl or —OCH₃.

In certain embodiments, the antisense strand of a single- ordouble-stranded siNA molecule of the invention may contain up to 9(e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9) additional chemically-modifiednucleotides with 2′-5′ internucleoside linkages, including but notlimited to a nucleotide of Formula III. In a further embodiment, theantisense strand of a single- or double-stranded siNA molecule of theinvention may contain up to 5 (e.g., 0, 1, 2, 3, 4 or 5) additionalchemically-modified nucleotides with 2′-5′ internucleoside linkages. Ina further embodiment, the antisense strand of a single- ordouble-stranded siNA molecule of the invention may contain up to 3(e.g., 0, 1, 2 or 3) additional chemically-modified nucleotides with2′-5′ internucleoside linkages. In another embodiment, the antisensestrand of a single- or double-stranded siNA molecule of the inventionmay contain one additional chemically-modified nucleotide with a 2′-5′internucleoside linkage.

Any of the above described modifications, or combinations thereof,including those in the references cited, can be applied to any of thesiNA molecules of the invention.

C. 5′ Modified Nucleotides

The instant invention features 5′ modified nucleotides that, whenincorporated into an siNA molecule of the invention, are located atnucleotide position 1 at the 5′ end of the antisense strand of themolecule, are linked to a nucleotide at position 2 of the antisensestrand through a 2′ internucleoside linkage, and may contain a modified5′ cap (i.e., other than a 5′ phosphate cap). These 5′ modifiednucleotides can be used as reagents to generate the siNA molecules ofthe invention.

In particular, the instant invention features 5′ modified nucleotideshaving the structure of Formula I:

wherein:

A is —C(R³)₂—, —C(R³)₂C(R³)₂— or —CR³═CR³—;

B is any heterocyclic base moiety;

D¹ and D^(1′) are independently selected from hydroxyl, —OR⁴, —SR⁴, or—N(R⁴)₂;

E and E′ are independently selected from O, S, —NR⁵, —N—N(R⁴)₂ or—N—OR⁴;

G¹ is hydroxyl or —OR⁶;

G^(1′) is hydroxyl, —OR⁶ or —N(R⁶)₂;

R¹ and R^(1′) are independently selected from H, hydroxyl, halogen, C₁₋₆alkyl, —OR⁷, —N(R⁷)₂, or together form ═O or ═CH₂;

R² is H, C₁₋₆ alkyl or C₂₋₆ alkenyl;

R₃ and R₅ are independently selected from H, hydroxyl, halogen, C₁₋₁₀alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, aryl,

wherein R′ is selected from H or C₁₋₄ alkyl (which is optionallysubstituted with one to three substituents independently selected from—SR¹¹, aryl, heteroaryl, amino, hydroxyl, oxo or —NH—C═(NH)NH₂, whereinthe aryl and heteroaryl are optionally substituted with hydroxyl), andR″ is selected from H, C₁₋₁₈ alkyl or aryl;

R⁴ is independently selected from H, C₁₋₁₀ alkyl, C₂₋₆ alkenyl, C₂₋₆alkynyl, aryl,

wherein R′ is selected from H or C₁₋₄ alkyl (which is optionallysubstituted with one to three substituents independently selected from—SR¹¹, aryl, heteroaryl, amino, hydroxyl, oxo or —NH—C═(NH)NH₂, whereinthe aryl and heteroaryl are optionally substituted with hydroxyl), andR″ is selected from H, C₁₋₁₈ alkyl or aryl;

R⁶ is independently C₁₋₆ alkyl, optionally substituted on the terminalcarbon atom with cyano or a protecting group;

R⁷ is independently selected from H, C₁₋₆ alkyl (which is optionallysubstituted with —OR⁸, —SR⁸, —N(R⁹)₂, (═O)—NR¹⁰ or from one to threehalogen), C₂₋₆ alkenyl, C₂₋₆ alkynyl, aryl,

wherein R′ is selected from H or C₁₋₄ alkyl (which is optionallysubstituted with one to three substituents independently selected from—SR¹¹, aryl, heteroaryl, amino, hydroxyl, oxo, —NH—C═(NH)NH₂, whereinthe aryl and heteroaryl are optionally substituted with hydroxyl), andR″ is selected from H, C₁₋₁₈ alkyl or aryl;

R⁸ is methyl, —CF₃, —N(R⁹)₂ or —CH₂—N(R⁹)₂;

R⁹ is independently selected from H or C₁₋₆ alkyl;

R¹⁰ is (R⁹)₂, —R⁹—(CH₂)₂—N(R⁹)₂ or —R⁹—C(═NR⁹)[N(R⁹)₂];

R¹¹ is H or C₁₋₄ alkyl; and,

r is 0 or 1.

In certain embodiments, R³ is independently selected from H, hydroxy, F,Cl, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, aryl,

wherein R′ is selected from H, C₁₋₄ alkyl (e.g., methyl, isopropyl,isobutyl, sec-butyl),

and R″ is selected from H, C₁₋₄ alkyl (e.g., methyl, ethyl, n-propyl,isopropyl) or aryl.

In certain embodiments, A is —CH₂CH₂— or —CH═CH—. In certainembodiments, A is CH═CH—.

In certain embodiments, B is selected from uracil, thymine, cytosine,5-methylcytosine, adenine or guanine. In certain embodiments, B isthymine.

In certain embodiments, D¹ and D^(1′) is independently selected fromhydroxyl, —OCH₃ or —OCH₂CH₃. In certain embodiments, D¹ and D^(1′) isindependently —OCH₂CH₃.

In certain embodiments, E is selected from O or S. In certainembodiments, E is O.

In certain embodiments, r is 1. In certain of these embodiments, where ris 1, G¹ and G^(1′) are hydroxyl, and E′ is O or S.

In certain embodiments, r is 0. In certain of these embodiments, whereinr is 0, G¹ is —O(CH₂)₂CN and G^(1′) is —N[CH(CH₃)₂]₂.

In certain embodiments, R¹ is H.

In certain embodiments, R^(1′) is H, halogen or —OR⁷. In certainembodiments, R^(1′) is halogen, —OCH₃, —OCH₂F, —OCHF₂, —OCF₃, —OCH₂CH₃,—O(CH₂)₂F, —OCH₂CHF₂, —OCH₂CF₃, —OCH₂—CH═CH₂, —O(CH₂)₂—OCH₃,—O(CH₂)₂—SCH₃, —O(CH₂)₂—OCF₃, —OCH₂C(═O)—N(H)CH₃ or—OCH₂C(═O)—N(H)—(CH₂)₂—N(CH₃)₂. In certain embodiments, R^(1′) is F or—OCH₃.

In certain embodiments, R² is H.

In certain embodiments, each of R¹, R^(1′) and R² is H.

D. Generation/Synthesis of siNA Molecules

The siNA molecules of the invention can be obtained using a number oftechniques known to those of skill in the art. For example the siNAmolecules can be chemically synthesized using protocols known in the art(for example, as described in: Caruthers et al., 1992, Methods inEnzymology 211, 3-19; Thompson et al., International PCT Publication No.WO 99/54459; Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684;Wincott et al., 1997, Methods Mol. Bio., 74, 59; Brennan et al., 1998,Biotechnol Bioeng., 61, 33-45; Brennan, U.S. Pat. No. 6,001,311; Usmanet al., 1987, J. Am. Chem. Soc., 109, 7845; and Scaringe et al., 1990,Nucleic Acids Res., 18, 5433). The syntheses of oligonucleotidesdescribed in the art makes use of common nucleic acid protecting andcoupling groups, such as dimethoxytrityl at the 5′-end andphosphoramidites at the 3′- or 2′-end. These syntheses can also be usedfor certain siNA molecules of the invention.

In certain embodiments, the siNA molecules of the invention aresynthesized, deprotected, and analyzed according to methods describedin, for example, U.S. Pat. Nos. 6,995,259, 6,686,463, 6,673,918,6,649,751, 6,989,442, and 7,205,399.

In a non-limiting synthesis example, small scale syntheses are conductedon a 394 Applied Biosystems, Inc. synthesizer using a 0.2 μmol scaleprotocol with a 2.5 min coupling step for 2′-O-methylated nucleotidesand a 45 second coupling step for 2′-deoxy nucleotides or2′-deoxy-2′-fluoro nucleotides.

Alternatively, the siNA molecules of the present invention can besynthesized separately and joined together post-synthetically, forexample, by ligation (e.g., Moore et al., 1992, Science 256, 9923;Draper et al., International PCT Publication No. WO 93/23569; Shabarovaet al., 1991, Nucleic Acids Research 19, 4247; Bellon et al., 1997,Nucleosides & Nucleotides, 16, 951; and Bellon et al., 1997,Bioconjugate Chem. 8, 204), or by hybridization following synthesisand/or deprotection.

E. Carrier/Delivery Systems

The siNA molecules of the invention are added directly to target cellsor tissues or complexed with various components (e.g., packaged withinliposomes; coupled with single chemical entity targeting moieties) fordelivery to target cells or tissues. Methods for the delivery of nucleicacid molecules are described in, for example, Akhtar et al., 1992,Trends Cell Bio., 2, 139; Delivery Strategies for AntisenseOligonucleotide Therapeutics, ed Akhtar, 1995; Maurer et al., 1999, Mol.Membr. Biol., 16, 129-140; Hofland and Huang, 1999, Handb. Exp.Pharmacol., 137, 165-192; and Lee et al., 2000, ACS Symp. Ser., 752,184-192. Beigelman et al., U.S. Pat. No. 6,395,713, and Sullivan et al.,PCT International application publication no. WO 94/02595, furtherdescribe the general methods for delivery of nucleic acid molecules.These protocols can be utilized for the delivery of virtually anynucleic acid molecule. Nucleic acid molecules can be administered tocells by a variety of methods known to those of skill in the art,including, but not restricted to, encapsulation in liposomes, byiontophoresis, or by incorporation into other vehicles, such asbiodegradable polymers, hydrogels, cyclodextrins (see for example,Gonzalez et al., 1999, Bioconjugate Chem., 10, 1068-1074; Wang et al.,International PCT Publication Nos. WO 03/47518 and WO 03/46185),poly(lactic-co-glycolic)acid (PLGA) and PLCA microspheres (see, e.g.,U.S. Pat. No. 6,447,796 and US Patent Application Publication No. US2002130430), biodegradable nanocapsules, and bioadhesive microspheres,or by proteinaceous vectors (e.g., O'Hare and Normand, International PCTPublication No. WO 00/53722).

In one aspect, the present invention provides carrier systems containingthe siNA molecules described herein. In some embodiments, the carriersystem is a lipid-based carrier system, cationic lipid, or liposomenucleic acid complexes, a liposome, a micelle, a virosome, a lipidnanoparticle or a mixture thereof. In other embodiments, the carriersystem is a polymer-based carrier system such as a cationicpolymer-nucleic acid complex. In additional embodiments, the carriersystem is a cyclodextrin-based carrier system such as a cyclodextrinpolymer-nucleic acid complex. In further embodiments, the carrier systemis a protein-based carrier system such as a cationic peptide-nucleicacid complex. In another embodiment, the carrier system is a lipidnanoparticle (“LNP”) formulation.

In certain embodiments, the siNA molecules of the invention areformulated with a lipid nanoparticle composition such as is described inU.S. Pat. No. 7,514,099 and U.S. Pat. No. 7,404,969, U.S. patentapplication Ser. Nos. 13/059,491, 13/390,702, 13/699,451, 13/701,636 and13/500,733, and PCT International Patent Appl. publication nos. WO2010/080724, WO 2011/090965, WO 2010/021865, WO 2010/042877, WO2010/105209, WO 2011/127255, WO 2012/040184, WO 2012/044638 and WO2011/022460.

In other embodiments, the invention features conjugates and/or complexesof siNA molecules of the invention. Such conjugates and/or complexes canbe used to facilitate delivery of siNA molecules into a biologicalsystem, such as a cell. The conjugates and complexes provided by theinstant invention have the potential of imparting therapeutic activityby transferring therapeutic compounds across cellular membranes,altering the pharmacokinetics, and/or modulating the localization ofnucleic acid molecules of the invention. Non-limiting, examples of suchconjugates are described in U.S. Pat. Nos. 8,137,695, 7,833,992,6,528,631, 6,335,434, 6,235,886, 6,153,737, 5,214,136, 5,138,045 and7,816,337, and U.S. patent application Ser. No. 10/201,394 (e.g.,CDM-LBA, CDM-Pip-LBA, CDM-PEG, CDM-NAG, etc.).

In various embodiments, polyethylene glycol (PEG) can be covalentlyattached to siNA compounds of the present invention. The attached PEGcan be any molecular weight, preferably from about 100 to about 50,000daltons (Da).

In yet other embodiments, the invention features compositions orformulations comprising surface-modified liposomes containing poly(ethylene glycol) lipids (PEG-modified, or long-circulating liposomes orstealth liposomes) and siNA molecules of the invention, such as isdisclosed in for example, International PCT Publication Nos. WO96/10391, WO 96/10390 and WO 96/10392.

In some embodiments, the siNA molecules of the invention can also beformulated or complexed with polyethyleneimine and derivatives thereof,such as polyethyleneimine-polyethyleneglycol-N-acetylgalactosamine(PEI-PEG-GAL) orpolyethyleneimine-polyethyleneglycol-tri-N-acetylgalactosamine(PEI-PEG-triGAL) derivatives. In one embodiment, the nucleic acidmolecules of the invention are formulated as described in U.S. PatentApplication publication no. 2003/0077829.

In other embodiments, siNA molecules of the invention are complexed withmembrane disruptive agents such as those described in U.S. Pat. No.6,835,393. In still other embodiments, the membrane disruptive agent oragents and the siNA molecule are also complexed with a cationic lipid orhelper lipid molecule, such as those lipids described in U.S. Pat. No.6,235,310.

In certain embodiments, siNA molecules of the invention are complexedwith delivery systems as described in U.S. Patent Applicationpublication nos. 2003/0077829; 2005/0287551; 2005/0191627; 2005/0118594;2005/0153919; 2005/0085486; and 2003/0158133; and International PCTpublication nos. WO 00/03683 and WO 02/087541.

In some embodiments, a liposomal formulation of the invention comprisesan siNA molecule of the invention (e.g., siNA) formulated or complexedwith compounds and compositions described in U.S. Pat. Nos. 6,858,224,6,534,484, 6,287,591, 6,835,395, 6,586,410, 6,858,225, 6,815,432,6,586,001, 6,120,798, 6,977,223, 6,998,115, 5,981,501, 5,976,567,5,705,385; and U.S. Patent Application publication nos. 2006/0019912,2006/0019258, 2006/0008909, 2005/0255153, 2005/0079212, 2005/0008689,2003/0077829, 2005/0064595, 2005/0175682, 2005/0118253, 2004/0071654,2005/0244504, 2005/0265961 and 2003/0077829.

F. Kits

The present invention also provides nucleic acids in kit form. The kitmay comprise a container. The kit typically contains a nucleic acid ofthe invention with instructions for its administration. In certaininstances, the nucleic acids may have a targeting moiety attached.Methods of attaching targeting moieties (e.g. antibodies, proteins) areknown to those of skill in the art. In certain instances, the nucleicacids are chemically modified. In other embodiments, the kit containsmore than one siNA molecule of the invention. The kits may comprise ansiNA molecule of the invention with a pharmaceutically acceptablecarrier or diluent. The kits may further comprise excipients.

G. Uses

siNA molecules of the present invention are useful to modulate (e g,inhibit) the expression of a target nucleic acid by an RNAi interferencemechanism. Thus, one aspect of the present invention relates to methodsof inhibiting gene expression by target-specific RNA interference in acell comprising contact said cell with an siNA molecule, or acomposition thereof, or the invention. The methods of inhibiting geneexpression of the invention may occur in vitro or in vivo. In certainembodiments, the cell is an animal cell (e.g., a mammalian cell). Incertain embodiments, the cell is a mammalian cell. In furtherembodiments, the mammalian cell is inside the animal.

The siNA molecules of the invention may be useful to regulate theexpression and/or activity of a target nucleic acid (i.e., a targetgene) and, thus, have the potential of being used in assays fordiagnostic purposes and/or in therapeutic regimens to treat one or moredisease states. For example, the one or more disease states having thepotential of being diagnosed and/or treated with an siNA of theinvention may be associated with the expression of a particular genetarget to which the siNA is directed. Thus, in this example, the siNAmolecules of the invention have the potential to degrade atarget-related mRNA, the gene expression of which is associated with aparticular disease state.

In one embodiment, inhibition of a disease may be evaluated by directlymeasuring the progress of the disease in a subject. For example, it mayalso be inferred through observing a change or reversal in a conditionassociated with the disease. siNA molecules of the invention also havethe potential of being used as a prophylaxis. Thus, use of the siNAmolecules and pharmaceutical compositions of the invention have thepotential of ameliorating, treating, preventing, and/or curing diseasesstates associated with regulation of gene expression of a particulartarget.

One aspect of the invention comprises a method of decreasing theexpression of a gene in a subject suffering from a condition or diseasewhich is mediated by the action, or by loss of action, of said targetgene, which method comprises administering to said subject an effectiveamount of an siNA molecule of the invention. In one embodiment, the siNAmolecule is directed to the gene (i.e., the target gene). In anotherembodiment, the siNA molecule is directed to a target within theexpression and/or activity pathway of gene (i.e., a pathway targetgene). In one embodiment, the subject is a human subject.

In one embodiment, a subject to which an siNA molecule of the invention,or composition thereof, may be administered is suffering from cancer ora condition associated with cancer. Thus, the siNA molecules of thepresent invention have the potential of being useful to treat cancer,including but not limited to the potential modulation of the metastasesof cancer cells and/or conditions associated with cancer. Examples ofcancers that have the potential of being treated according to thisaspect of the invention may include bladder cancer, bladder transitionalcell carcinoma, urothelial carcinoma, brain cancer, gliomas,astrocytomas, breast cancer, breast carcinoma, cervical cancer,colorectal cancer, rectal cancer, colorectal carcinoma, colon cancer,hereditary nonpolyposis colorectal cancer, endometrial carcinoma,esophageal cancer, esophageal squamous cell carcinoma, ocular melanoma,uveal melanoma, intraocular melanoma, primary intraocular lymphoma,renal cell carcinoma, clear cell renal cell carcinoma, papillary renalcell carcinoma, leukemia, acute lymocytic leukemia (ALL), acute myeloidleukemia (AML), chronic myeloid (CML), chronic myelomonocytic (CMML),liver cancer, hepatoma, hepatocellular carcinoma, cholangiocarcinoma,lung cancer, small cell lung cancer (SCLC), non-small cell lung cancer(NSCLC), Non-Hodgkin lymphoma, B-cell lymphomas, T-cell lymphomas,precursor T-lymphoblastic lymphoma/leukemia, multiple myeloma, ovariancarcinoma, pancreatic cancer, pancreatic ductal adenocarcinoma, prostatecancer, prostate adenocarcinoma, rhabdomyosarcoma, embryonalrhabdomyosarcoma, skin cancer, melanoma, malignant melanoma, cutaneousmelanoma, mucosal melanoma, small intestine carcinomas, stomach cancer,gastric carcinoma, testicular cancer, testicular seminomas, testicularnon-seminomas, thyroid cancer, papillary thyroid carcinoma, andpapillary adenocarcinomas.

siNA molecules of the invention have the potential of being useful asreagents in ex vivo applications. For example, siNA molecules may beintroduced into tissue or cells that are transplanted into a subject fora potential therapeutic effect. The cells and/or tissue can be derivedfrom an organism or subject that later receives the explant, or can bederived from another organism or subject prior to transplantation. Inthis context, the siNA molecules may modulate the expression of one ormore genes in the cells or tissue, such that the cells or tissue obtaina desired phenotype or are able to perform a function when transplantedin vivo.

H. Pharmaceutical Compositions

The siNA molecules of the instant invention have the potential ofproviding useful reagents for use in methods related to a variety oftherapeutic, prophylactic, veterinary, diagnostic, target validation,genomic discovery, genetic engineering, and pharmacogenomicapplications.

1. Formulations

The present invention provides for pharmaceutical compositions of thesiNA molecules described, i.e., compositions in a pharmaceuticallyacceptable carrier or diluent. These formulations or compositions cancomprise a pharmaceutically acceptable carrier or diluent as isgenerally known in the art.

The siNA molecules of the invention are preferably formulated aspharmaceutical compositions prior to administering to a subject,according to techniques known in the art. Pharmaceutical compositions ofthe present invention are characterized as being at least sterile andpyrogen-free. Methods for preparing pharmaceutical compositions of theinvention are within the skill in the art for example as described inRemington's Pharmaceutical Science, 17^(th) ed., Mack PublishingCompany, Easton, Pa. (1985).

In some embodiments, pharmaceutical compositions of the invention (e.g.,siNA and/or LNP formulations thereof) further comprise conventionalpharmaceutical excipients and/or additives. Suitable pharmaceuticalexcipients include preservatives, flavoring agents, stabilizers,antioxidants, osmolality adjusting agents, buffers, and pH adjustingagents. Suitable additives include physiologically biocompatible buffers(e.g., trimethylamine hydrochloride), addition of chelants (such as,e.g., DTPA or DTPA-bisamide) or calcium chelate complexes (e.g. calciumDTPA, CaNaDTPA-bisamide), or, optionally, additions of calcium or sodiumsalts (e.g., calcium chloride, calcium ascorbate, calcium gluconate orcalcium lactate). In addition, antioxidants and suspending agents can beused.

Non-limiting examples of various types of formulations for localadministration include ointments, lotions, creams, gels, foams,preparations for delivery by transdermal patches, powders, sprays,aerosols, capsules or cartridges for use in an inhaler or insufflator ordrops (e.g., eye or nose drops), solutions/suspensions for nebulization,suppositories, pessaries, retention enemas and chewable or suckabletablets or pellets (e.g., for the treatment of aphthous ulcers) orliposome or microencapsulation preparations.

Ointments, creams and gels, can, for example, can be formulated with anaqueous or oily base with the addition of suitable thickening and/orgelling agent and/or solvents. Non-limiting examples of such bases canthus, e.g., include water and/or an oil (such as liquid paraffin) or avegetable oil (such as arachis oil or castor oil), or a solvent (such aspolyethylene glycol). Various thickening agents and gelling agents canbe used depending on the nature of the base. Non-limiting examples ofsuch agents include soft paraffin, aluminum stearate, cetostearylalcohol, polyethylene glycols, woolfat, beeswax, carboxypolymethyleneand cellulose derivatives, and/or glyceryl monostearate and/or non-ionicemulsifying agents.

In one embodiment, lotions can be formulated with an aqueous or oilybase and will in general also contain one or more emulsifying agents,stabilizing agents, dispersing agents, suspending agents or thickeningagents.

In one embodiment, powders for external application can be formed withthe aid of any suitable powder base, e.g., talc, lactose or starch.Drops can be formulated with an aqueous or non-aqueous base alsocomprising one or more dispersing agents, solubilizing agents,suspending agents or preservatives.

Compositions intended for oral use can be prepared according to anymethod known to the art for the manufacture of pharmaceuticalcompositions, and such compositions can contain one or more suchsweetening agents, flavoring agents, coloring agents or preservativeagents in order to provide pharmaceutically elegant and palatablepreparations. Tablets contain the active ingredient in admixture withnon-toxic pharmaceutically acceptable excipients that are suitable forthe manufacture of tablets. These excipients can be, e.g., inertdiluents (such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate), granulating and disintegrating agents(e.g., corn starch, or alginic acid), binding agents (e.g., starch,gelatin or acacia), and lubricating agents (e.g., magnesium stearate,stearic acid or talc). The tablets can be uncoated or they can be coatedby known techniques. In some cases such coatings can be prepared byknown techniques to delay disintegration and absorption in thegastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonosterate or glyceryl distearate can be employed.

Formulations for oral use can also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent,e.g., calcium carbonate, calcium phosphate or kaolin, or as soft gelatincapsules wherein the active ingredient is mixed with water or an oilmedium, e.g., peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active materials in a mixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, e.g., sodium carboxymethylcellulose,methylcellulose, hydropropyl-methylcellulose, sodium alginate,polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing orwetting agents can be a naturally-occurring phosphatide, e.g., lecithin,or condensation products of an alkylene oxide with fatty acids, e.g.,polyoxyethylene stearate; or condensation products of ethylene oxidewith long chain aliphatic alcohols, e.g., heptadecaethyleneoxycetanol,or condensation products of ethylene oxide with partial esters derivedfrom fatty acids and a hexitol such as polyoxyethylene sorbitolmonooleate, or condensation products of ethylene oxide with partialesters derived from fatty acids and hexitol anhydrides, e.g.,polyethylene sorbitan monooleate. The aqueous suspensions can alsocontain one or more preservatives, e.g. ethyl, or n-propylp-hydroxybenzoate, one or more coloring agents, one or more flavoringagents, and one or more sweetening agents, such as sucrose or saccharin.

Oily suspensions can be formulated by suspending the active ingredientsin a vegetable oil, e.g. arachis oil, olive oil, sesame oil or coconutoil, or in a mineral oil such as liquid paraffin. The oily suspensionscan contain a thickening agent, e.g. beeswax, hard paraffin or cetylalcohol. Sweetening agents and flavoring agents can be added to providepalatable oral preparations. These compositions can be preserved by theaddition of an anti-oxidant such as ascorbic acid

Pharmaceutical compositions of the invention can also be in the form ofoil-in-water emulsions. The oily phase can be a vegetable oil or amineral oil or mixtures of these. Suitable emulsifying agents can benaturally-occurring gums, e.g. gum acacia or gum tragacanth,naturally-occurring phosphatides, e.g. soy bean, lecithin, and esters orpartial esters derived from fatty acids and hexitol, anhydrides, forexample sorbitan monooleate, and condensation products of the saidpartial esters with ethylene oxide, e.g. polyoxyethylene sorbitanmonooleate. The emulsions can also contain sweetening and flavoringagents.

Syrups and elixirs can be formulated with sweetening agents, e.g.glycerol, propylene glycol, sorbitol, glucose or sucrose. Suchformulations can also contain a demulcent, a preservative, and flavoringand coloring agents. The pharmaceutical compositions can be in the formof a sterile injectable aqueous or oleaginous suspension. Thissuspension can be formulated according to the known art using thosesuitable dispersing or wetting agents and suspending agents that havebeen mentioned above. The sterile injectable preparation can also be asterile injectable solution or suspension in a non-toxic parentallyacceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that can beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose, any bland fixed oilcan be employed including synthetic mono- or diglycerides. In addition,fatty acids such as oleic acid find use in the preparation ofinjectables.

The siNA molecules of the invention can take the form of suppositories,e.g., for rectal administration of the drug. These compositions can beprepared by mixing the drug with a suitable non-irritating excipientthat is solid at ordinary temperatures but liquid at the rectaltemperature and will therefore melt in the rectum to release the drug.Such materials include cocoa butter and polyethylene glycols.

siNA molecules of the invention can be formulated in a sterile mediumfor parenteral administration. The molecule, depending on the vehicleand concentration used, can either be suspended or dissolved in thevehicle. Advantageously, adjuvants such as local anesthetics,preservatives and buffering agents can be dissolved in the vehicle.

In other embodiments, siNA molecule formulations provided herein for usein pulmonary delivery further comprise one or more surfactants. Suitablesurfactants or surfactant components for enhancing the uptake of thecompositions of the invention include synthetic and natural as well asfull and truncated forms of surfactant protein A, surfactant protein B,surfactant protein C, surfactant protein D and surfactant Protein E,di-saturated phosphatidylcholine (other than dipalmitoyl),dipalmitoylphosphatidylcholine, phosphatidylcholine,phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine,phosphatidylserine; phosphatidic acid, ubiquinones,lysophosphatidylethanolamine, lysophosphatidylcholine,palmitoyl-lysophosphatidylcholine, dehydroepiandrosterone, dolichols,sulfatidic acid, glycerol-3-phosphate, dihydroxyacetone phosphate,glycerol, glycero-3-phosphocholine, dihydroxyacetone, palmitate,cytidine diphosphate (CDP) diacylglycerol, CDP choline, choline, and/orcholine phosphate; as well as natural and artificial lamellar bodieswhich are the natural carrier vehicles for the components of surfactant,omega-3 fatty acids, polyenic acid, polyenoic acid, lecithin, palmitinicacid, non-ionic block copolymers of ethylene or propylene oxides,polyoxypropylene, monomeric and polymeric, polyoxyethylene, monomericand polymeric, poly (vinyl amine) with dextran and/or alkanoyl sidechains, Brij 35, Triton X-100 and synthetic surfactants ALEC, Exosurf,Survan and Atovaquone, among others. These surfactants can be usedeither as single or part of a multiple component surfactant in aformulation, or as covalently bound additions to the 5′ and/or 3′ endsof the nucleic acid component of a pharmaceutical composition herein.

In one embodiment, the siNA molecules of the invention can be formulatedfor administration via pulmonary delivery, such as by inhalation of anaerosol or spray dried formulation administered by an inhalation deviceor nebulizer, providing rapid local uptake of the nucleic acid moleculesinto relevant pulmonary tissues. Solid particulate compositionscontaining respirable dry particles of micronized nucleic acidcompositions can be prepared by grinding dried or lyophilized nucleicacid compositions, and then passing the micronized composition through,for example, a 400 mesh screen to break up or separate out largeagglomerates. A solid particulate composition comprising the siNAcompositions of the invention can optionally contain a dispersant whichserves to facilitate the formation of an aerosol as well as othertherapeutic compounds. A suitable dispersant is lactose, which can beblended with the nucleic acid compound in any suitable ratio, such as a1 to 1 ratio by weight.

Spray compositions comprising siNA molecules of the invention can, forexample, be formulated as aqueous solutions or suspensions or asaerosols delivered from pressurized packs, such as a metered doseinhaler, with the use of a suitable liquefied propellant. In oneembodiment, aerosol compositions of the invention suitable forinhalation can be either a suspension or a solution and generallycontain an siNA molecule of the invention and a suitable propellant suchas a fluorocarbon or hydrogen-containing chlorofluorocarbon or mixturesthereof, particularly hydrofluoroalkanes, especially1,1,1,2-tetrafluoroethane, 1,1,1,2,3,3,3-heptafluoro-n-propane or amixture thereof. The aerosol composition can optionally containadditional formulation excipients well known in the art such assurfactants. Non-limiting examples include oleic acid, lecithin or anoligolactic acid or derivative such as those described in WO94/21229 andWO98/34596 and co-solvents for example ethanol. In one embodiment, apharmaceutical aerosol formulation of the invention comprising acompound of the invention and a fluorocarbon or hydrogen-containingchlorofluorocarbon or mixtures thereof as propellant, optionally incombination with a surfactant and/or a co-solvent.

The aerosol formulations of the invention can be buffered by theaddition of suitable buffering agents.

Aerosol formulations can include optional additives includingpreservatives if the formulation is not prepared sterile. Non-limitingexamples include, methyl hydroxybenzoate, anti-oxidants, flavorings,volatile oils, buffering agents and emulsifiers and other formulationsurfactants. In one embodiment, fluorocarbon or perfluorocarbon carriersare used to reduce degradation and provide safer biocompatiblenon-liquid particulate suspension compositions of the invention (e.g.,siNA and/or LNP formulations thereof). In another embodiment, a devicecomprising a nebulizer delivers a composition of the invention (e.g.,siNA and/or LNP formulations thereof) comprising fluorochemicals thatare bacteriostatic thereby decreasing the potential for microbial growthin compatible devices.

Capsules and cartridges comprising the composition of the invention foruse in an inhaler or insufflator, of for example gelatin, can beformulated containing a powder mix for inhalation of a compound of theinvention and a suitable powder base such as lactose or starch. In oneembodiment, each capsule or cartridge contains an siNA molecule of theinvention and one or more excipients. In another embodiment, thecompound of the invention can be presented without excipients such aslactose.

The siNA molecules can also be formulated as a fluid formulation fordelivery from a fluid dispenser, such as those described and illustratedin WO05/044354.

2. Combinations

The siNA molecules and pharmaceutical formulations according to theinvention can be administered to a subject alone or used in combinationwith one or more other therapies, including known therapeutic agents,treatments, or procedures to prevent or treat diseases, disorders,conditions, and traits. A person of ordinary skill in the art would beable to discern which combinations of therapeutic agents would be usefulbased on the particular characteristics of the drug components and thedisease indication/state involved, including but not limited cancer.

A combination can conveniently be presented for use in the form of apharmaceutical composition, wherein the pharmaceutical compositioncomprises a combination that includes an siNA molecule of the invention,a pharmaceutically acceptable diluent or carrier, and one or moreadditional therapeutic agents. Alternatively, the individual componentsof such combinations can be administered either sequentially orsimultaneously in separate or combined pharmaceutical formulations.

I. Administration

Compositions or formulations may be administered in a variety of ways.In certain embodiments, the administration of an siNA molecule is vialocal administration or systemic administration, either alone as amonotherapy or in combination with additional therapies described hereinor as are known in the art.

Local administration can include, for example, inhalation, nebulization,catheterization, implantation, direct injection, dermal/transdermalapplication, patches, stenting, ear/eye drops, or portal veinadministration to relevant tissues, or any other local administrationtechnique, method or procedure, as is generally known in the art.Systemic administration can include, for example, pulmonary (inhalation,nebulization etc.) intravenous, subcutaneous, intramuscular,catheterization, nasopharyngeal, transdermal, or oral/gastrointestinaladministration as is generally known in the art. Further non-limitingexamples of administration methods of the invention include buccal,sublingual, parenteral (i.e., intraarticularly, intravenously,intraperitoneally, subcutaneously, or intramuscularly), local rectaladministration or other local administration. In one embodiment, thecomposition of the invention can be administered by insufflation andinhalation.

In one embodiment, the siNA molecules of the invention and formulationsor compositions thereof are administered to the liver via methodsgenerally known in the art (see, e.g., Wen et al., 2004, World JGastroenterol., 10, 244-9; Murao et al., 2002, Pharm Res., 19, 1808-14;Liu et al., 2003, gene Ther., 10, 180-7; Hong et al., 2003, J PharmPharmacol., 54, 51-8; Herrmann et al., 2004, Arch Virol., 149, 1611-7;and Matsuno et al., 2003, gene Ther., 10, 1559-66).

In one embodiment, the invention features the use of methods to deliverthe siNA molecules of the instant invention and compositions thereof tohematopoietic cells, including monocytes and lymphocytes. These methodsare described in detail by Hartmann et al., 1998, J. Pharmacol. Exp.Ther., 285(2), 920-928; Kronenwett et al., 1998, Blood, 91(3), 852-862;Filion and Phillips, 1997, Biochim. Biophys. Acta., 1329(2), 345-356; Maand Wei, 1996, Leuk. Res., 20(11/12), 925-930; and Bongartz et al.,1994, Nucleic Acids Research, 22(22), 4681-8.

In one embodiment, the siNA molecules of the invention and formulationsor compositions thereof are administered directly or topically (e.g.,locally) to the dermis or follicles via methods generally known in theart (see, e.g., Brand, 2001, Curr. Opin. Mol. Ther., 3, 244-8; Regnieret al., 1998, J. Drug Target, 5, 275-89; Kanikkannan, 2002, BioDrugs,16, 339-47; Wraight et al., 2001, Pharmacol. Ther., 90, 89-104; andPreat and Dujardin, 2001, STP PharmaSciences, 11, 57-68). In oneembodiment, the siNA molecules of the invention and formulations orcompositions thereof are administered directly or topically using ahydroalcoholic gel formulation comprising an alcohol (e.g., ethanol orisopropanol), water, and optionally including additional agents suchisopropyl myristate and carbomer 980. In other embodiments, the siNAmolecules and compositions are administered topically to the nasalcavity. Topical preparations can be administered by one or moreapplications per day to the affected area. Continuous or prolongeddelivery can be achieved by an adhesive reservoir system.

In one embodiment, an siNA molecule of the invention or a compositionthereof is administered iontophoretically, for example to a particularorgan or compartment (e.g., the eye, back of the eye, heart, liver,kidney, bladder, prostate, tumor, CNS etc.). Non-limiting examples ofiontophoretic delivery are described in, for example, WO 03/043689 andWO 03/030989, which are incorporated by reference in their entiretiesherein.

In some embodiments, the pharmaceutical compositions are administeredintravenously or intraperitoneally by a bolus injection (see, e.g., U.S.Pat. No. 5,286,634). Lipid nucleic acid particles can be administered bydirect injection at the site of disease or by injection at a site distalfrom the site of disease (see, e.g., Culver, HUMAN GENE THERAPY, MaryAnnLiebert, Inc., Publishers, New York. pp. 70-71(1994)).

For therapeutic applications, a pharmaceutically effective dose of thesiNA molecules or pharmaceutical compositions of the invention isadministered to the subject. A pharmaceutically effective dose is thatdose required to prevent, inhibit the occurrence, or treat (alleviate asymptom to some extent, preferably all of the symptoms) a disease state.One skilled in the art can readily determine a therapeutically effectivedose of an siNA molecule of the invention to be administered to a givensubject, e.g., by taking into account factors, such as the size andweight of the subject, the extent of the disease progression orpenetration, the age, health, and sex of the subject, the route ofadministration, and whether the administration is regional or systemic.Generally, an amount between 0.1 μg/kg and 100 mg/kg body weight/day ofactive ingredients is administered dependent upon potency of thenegatively charged polymer. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient. ThesiNA molecules of the invention can be administered in a single dose orin multiple doses.

siNA molecules of the instant invention can be administered oncemonthly, once weekly, once daily (QD), or divided into multiple monthly,weekly, or daily doses, such as, for example, twice daily (BID), threetimes daily (TID), once every two weeks. Thus, administration can beaccomplished via single or divided doses. Persons of ordinary skill inthe art can easily estimate repetition rates for dosing based onmeasured residence times and concentrations of the drug in bodily fluidsor tissues.

In addition, the administration can be continuous, e.g., every day, orintermittently. For example, intermittent administration of an siNAmolecule of the instant invention may be administration one to six daysper week, administration in cycles (e.g., daily administration for twoto eight consecutive weeks, then a rest period with no administrationfor up to one week), or administration on alternate days.

The compositions of the present invention, either alone or incombination with other suitable components, can be made into aerosolformulations (i.e., they can be “nebulized”) to be administered viainhalation (e.g., intranasally or intratracheally) (see, Brigham et al.,Am. J. Sci., 298:278 (1989)). Aerosol formulations can be placed intopressurized acceptable propellants, such as dichlorodifluoromethane,propane, nitrogen, and the like.

The aerosol compositions of the present invention can be administeredinto the respiratory system as a formulation that includes particles ofrespirable size, e.g. particles of a size sufficiently small to passthrough the nose, mouth and larynx upon inhalation and through thebronchi and alveoli of the lungs. In general, respirable particles rangefrom about 0.5 to 10 microns in size. In one embodiment, the particulaterange can be from 1 to 5 microns. In another embodiment, the particulaterange can be from 2 to 3 microns. Particles of non-respirable size whichare included in the aerosol tend to deposit in the throat and beswallowed, and the quantity of non-respirable particles in the aerosolis thus minimized. For nasal administration, a particle size in therange of 10-500 um is preferred to ensure retention in the nasal cavity.

In some embodiments, an siNA composition of the invention isadministered topically to the nose for example, for the treatment ofrhinitis, via pressurized aerosol formulations, aqueous formulationsadministered to the nose by pressurized pump or by nebulization.

Solid particle aerosols comprising an siNA molecule or formulation ofthe invention and surfactant can be produced with any solid particulateaerosol generator. One type of solid particle aerosol generator usedwith the siNA molecules of the invention is an insufflator. A secondtype of illustrative aerosol generator comprises a metered dose inhaler(“MDI”). MDIs containing siNA molecules or formulations taught hereincan be prepared by methods of the art (for example, see Byron, above andWO96/32099).

Thus, in certain embodiments of the invention, nebulizer devices areused in applications for conscious, spontaneously breathing subjects,and for controlled ventilated subjects of all ages. The nebulizerdevices can be used for targeted topical and systemic drug delivery tothe lung. In one embodiment, a device comprising a nebulizer is used todeliver an siNA molecule or formulation of the invention locally to lungor pulmonary tissues. In another embodiment, a device comprising anebulizer is used to deliver an siNA molecule or formulation of theinvention systemically.

J. Other Applications/Uses of siNA Molecules of the Invention

The siNA molecules of the invention can also be used for diagnosticapplications, research applications, and/or manufacture of medicaments.

In one aspect, the invention features a method for diagnosing a disease,trait, or condition in a subject comprising administering to the subjecta composition of the invention under conditions suitable for thediagnosis of the disease, trait, or condition in the subject.

In another embodiment, the invention comprises use of a double-strandednucleic acid according to the invention for use in the manufacture of amedicament. In an embodiment, the medicament is for use in treating acondition that is mediated by the action, or by loss of action, of agene or protein.

EXAMPLES

Examples provided are intended to assist in a further understanding ofthe invention. Particular materials employed, species and conditions areintended to be illustrative of the invention and not limiting of thereasonable scope thereof. Certain starting materials and reagents areeither commercially available or known in the chemical scientific orpatent literature.

The abbreviations used herein have the following tabulated meanings (seeTable 1). Abbreviations not tabulated below have their meanings ascommonly used unless specifically stated otherwise.

TABLE 1 Abbreviations TBDMSC1 t-butyldimethylchlorosilane DMTrCl4,4′-dimethyloxytrityl chloride DCM dichloromethane DMAP4-dimethylaminopyridine EtOAc ethylacetate Na₂SO₄ sodium sulfate AIBNazobisisobutyronitrile SnH(Bu)₃ tri-n-butyltin hydride DCAAdichloroacetic acid MgSO₄ magnesium sulfate TFA trifluoroacetic acidDMSO dimethyl sulfoxide DCC N,N′-dicyclohexylcarbodiimide THFtetrahydrofuran KOtBu potassium t-butoxide NH₄Cl ammonium chloride ACNor MeCN acetonitrile DIPEA N,N′-diisopropylethylamine NaHCO₃ sodiumbicarbonate NaOH sodium hydroxide TBSCl tert-butylchlorodimethylsilaneTBAF tetrabutylammoniumfluoride Et₃N triethylamine

General Schemes

Generic Scheme 1 can be used to generate 5′ modified nucleotides ofFormula I, wherein A is —C(R³)₂C(R³)₂— and R³ is H. B can be anyheterocyclic base moiety (e.g., uracil, thymine, cytosine,5-methylcytosine, guanine etc.). These phosphoramidites can be used tomake siNA molecules of the invention.

Generic Scheme 2 can be used to generate 5′ modified nucleotides ofFormula I, wherein A is —C(R³)₂C(R³)₂— and R³ is OAc. B can be anyheterocyclic base moiety (e.g., uracil, thymine, cytosine,5-methylcytosine, guanine etc.). These phosphoramidites can be used tomake siNA molecules of the invention.

Generic Scheme 3 can be used to generate 5′ modified nucleotides ofFormula I, wherein A is —C(R³)₂C(R³)₂— and R³ is OAc or H. B can be anyheterocyclic base moiety (e.g., uracil, thymine, cytosine,5-methylcytosine, guanine etc.). These phosphoramidites can be used tomake siNA molecules of the invention.

Generic Scheme 4 can be used to generate 5′ modified nucleotides ofFormula I, wherein A is —C(R³)₂C(R³)₂— and R³ is —OAc, —NHAc or F. B canbe any heterocyclic base moiety (e.g., uracil, thymine, cytosine,5-methylcytosine, guanine etc.). These phosphoramidites can be used tomake siNA molecules of the invention.

Generic Scheme 5 can be used to generate 5′ modified nucleotides ofFormula I, wherein A is —C(R³)₂C(R³)₂— and R³ is —OC(O)iPr, —NHC(O)CF₃or F. B can be any heterocyclic base moiety (e.g., uracil, thymine,cytosine, 5-methylcytosine, guanine etc.). These phosphoramidites can beused to make siNA molecules of the invention.

Generic Scheme 6 can be used to generate 5′ modified nucleotides ofFormula I, wherein A is —C(R³)₂C(R³)₂— and R³ is —OTBS, —NHC(O)CF₃ or F.B can be any heterocyclic base moiety (e.g., uracil, thymine, cytosine,5-methylcytosine, guanine etc.). These phosphoramidites can be used tomake siNA molecules of the invention.

Generic Scheme 7 can be used to generate 5′ modified nucleotides ofFormula I, wherein A is —C(R³)₂C(R³)₂— and R³ is NHC(O)CF₃ or F. B canbe any heterocyclic base moiety (e.g., uracil, thymine, cytosine,5-methylcytosine, guanine etc.). These phosphoramidites can be used tomake siNA molecules of the invention.

Generic Scheme 8 can be used to generate 5′ modified nucleotides ofFormula I, wherein A is —OC(R³)₂—, R³ is H, and R¹ and R^(1′) are H,—OMe, —OH or together ═CH₂. These phosphoramidites can be used to makesiNA molecules of the invention.

Example 1: Synthesis of Compound 8

Compound 8 is a phosphoramidite that can be used to generate single-and/or double-stranded siNA molecules comprising a “vinylP3dT” or“vinylP3dTs” nucleotide at the first nucleotide position of the molecule(position 1, which includes a 5′ cap; also referred to herein as“5′-position 1”). The chemical structure for vinylP3dT and vinylP3dTscan be found within Table 14, infra. Generally, a similar synthesisprocedure can be used to make phosphoramidites that are used to generatesingle and/or double-stranded siNA molecules comprising vinylIP3dX orvinylIP3dXs at position 1, wherein X is any heterocyclic base moiety.

1.1. Preparation of Compound 2

To a solution of 5-methyluridine (50 g, 194 mmol) 1 in anhydrouspyridine (300 mL) at rt was added 4,4′-dimethyloxytriyl chloride (66.9g, 197 mmol) over 5 minutes. The resulting mixture was stirred at rt for2 h followed by the addition of t-butyldimethylchlorosilane (30.3 g, 201mmol) over 10 min. The resulting mixture was stirred for 12 h and1-methylimidazole (3.09 mL, 38.7 mmol) and excess oft-butyldimethylchlorosiliane (20.4 g, mmol) were added. The resultingmixture was stirred for 8 h and then filtered. The filtrate wasconcentrated, diluted with EtOAC, washed with brine twice, dried overNa₂SO₄ and concentrated. The crude product was loaded onto a silicacolumn (pre-equilibrated with Hex). It was eluted with 0 to 40% EtOAc inHex to give the DMTr-TBDMS protected 5-methyluridine analog 2 as a whitefoam. ¹H NMR (400 MHz, CD₃CN) δ=9.10 (s, 1H); 7.50 (d, J=1.2 Hz, 1H);7.49-7.42 (m, 2H); 7.34-7.30 (m, 6H); 7.27-7.23 (m, 1H); 6.90-6.86 (m,4H); 5.89 (d, J=5.6 Hz, 1H); 4.42 (t, J=5.3 Hz, 1H); 4.23 (dd, J=8.6,0.8 Hz, 1H); 4.07-4.03 (m, 1H); 3.77 (s, 6H); 3.36-3.29 (m, 2H); 3.12(d, J=4.8 Hz 1H); 1.41 (d, J=1.1 Hz, 3H); 0.91 (s, 9H); 0.13 (d, J=11.4Hz, 6H)

1.2. Preparation of Compound 3

To a solution of compound 2 (28.16 g, 41.7 mmol) in anhydrous DCM (170mL) at rt was added 4-dimethylaminopyridine (15.29 g, 125 mmol). Theresulting mixture was stirred until all solids fully dissolved. Phenylchlorothionocarbonate (10.13 mL, 75 mmol) was added to the reactionmixture over 5 minutes. The resulting mixture was stirred at rt for 24h. The crude reaction was concentrated under reduced pressure. The crudeproduct was dissolved in minimal amount of CH₂Cl₂ and loaded to silicacolumn (pre-equilibriated with Hex). It was eluted with 0 to 30% EtOAcin Hex to give compound 3 as off-white foam. ¹H NMR (400 MHz, CDCl₃)δ=8.39 (s, 1H); 7.68 (d, J=1.1 Hz, 1H); 7.46-7.42 (m, 4H); 7.33-7.29 (m,8H); 7.09-7.07 (m, 2H); 6.87-6.84 (m, 4H); 6.15 (d, J=5.8 Hz, 1H); 6.00(dd, J=5.0, 3.4 Hz, 1H); 4.74 (t, J=5.5 Hz, 1H); 4.43-4.41 (m, 1H); 3.80(d, J=1.7 Hz, 6H); 3.56-3.49 (m, 2H); 1.45 (d, J=0.9 Hz, 3H); 0.93 (s,9H); 0.15 (d, J=13.6 Hz, 6H).

1.3. Preparation of Compound 4

Compound 3 (18.44 g, 22.74 mmol) and AIBN (3.88 g, 23.65 mmol) weredissolved in anhydrous, degassed toluene (180 mL). To the resultingmixture was added Tri-n-butyltin hydride (11.7 mL, 43.7 mmol) over 3minutes at rt. The resulting mixture was heated to 95° C. for 1 hour,cooled to rt, concentrated, and loaded to a silica column. The crudeproduct was eluted with 0 to 40% EtOAc in Hex to give compound 4 as awhite foam. ¹H NMR (400 MHz, CDCl₃) δ=8.44 (s, 1H); 7.72 (d, J=1.2 Hz,1H); 7.45-7.42 (m, 2H); 7.35-7.25 (m, 7H); 6.87-6.83 (m, 4H); 5.76 (d,J=1.52 Hz, 1H); 4.58-4.54 (m, 1H); 4.47-4.46 (m, 1H); 3.80 (d, J=0.4 Hz,6H); 3.61 (dd, J=10.9, 2.2 Hz, 1H); 3.31-3.22 (m, 1H); 2.24-2.17 (m,1H); 1.89-1.84 (m, 1H); 1.40 (d, J=1.0 Hz, 3H); 0.90 (s, 9H); 0.17 (d,J=22.0 Hz, 6H).

1.4. Preparation of Compound 5

To a solution of compound 4 (4.9 g, 7.44 mmol) in anhydrous DCM (50 mL)was added dichloroacetic acid (1.66 mL, 20.08 mmol) and dodecanethiol(1.781 mL, 7.44 mmol). The resulting mixture was stirred at rt for 25min followed by the addition of 0.6 M sodium bicarbonate solution (37.2mL, 22.31 mmol). The aqueous layer was extracted once with DCM, driedover MgSO₄ and concentrated under reduced pressure. The crude productwas loaded onto a silica column (pre-equilibrated with Hex). It waseluted with 0 to 50% EtOAc in Hex to give compound 5 as a colorless oil.¹H NMR (400 MHz, CDCl₃) δ=8.95 (s, 1H); 7.61 (s, 1H); 5.62 (d, J=1.9 Hz,1H); 4.50-4.47 (m, 2H); 4.09 (dd, J=12.2, 2.24 Hz, 1H); 3.74 (dd,J=12.2, 3.1 Hz, 1H); 2.19-2.12 (m, 1H); 1.89 (s, 3H); 1.87-1.81 (m, 1H);0.88 (s, 9H); 0.11 (d, J=13.2 Hz, 6H).

1.5. Preparation of Compound 6a

To a solution of compound 5 (2.16 g, 6.07 mmol) and pyridine-TFA (0.586g, 3.04 mmol) in anhydrous DMSO (4 mL) at 0-5° C. was added DCC (1.0 Msolution in DCM, 18.22 mL, 18.22 mmol) over 3 min. The resulting mixturewas stirred at rt for 1 h. The crude aldehyde solution was used in thefollowing reaction without further purification. In a separate reactionvessel, tetraethyl methylenediphosphonate (2.411 mL, 9.70 mmol) wasdissolved in anhydrous THF (10 mL) and cooled to 0° C., followed byaddition of potassium t-butoxide (1.0 M, 9.10 mL) and agitated at rt for30 min and then cooled to 0° C. The solution containing aldehyde wasadded to this solution at 0° C. and agitated for 15 min. The resultingmixture was quenched into a mixture of brine and EtOAC, and pH adjustedto ˜8 with NH₄Cl. The organic layer was washed twice with brine, driedover MgSO₄ and concentrated under reduced pressure. The crude productwas loaded onto a silica column (pre-equilibrated with Hex). It waseluted with 0 to 80% EtOAc in Hex to give 6a as white foam. ¹H NMR (400MHz, CDCl₃) δ=8.84 (s, 1H); 7.13 (d, J=1.2 Hz, 1H); 6.97 (dt, J=22.0,4.3 Hz, 1H); 6.12 (dt, J=18.9, 1.76 Hz, 1H); 5.77 (d, J=0.6 HZ, 1H);5.01-4.97 (m, 1H); 4.39 (d, J=4.2 Hz, 1H); 4.17-4.08 (m, 4H); 4.09-4.05(m, 1H); 2.11-2.06 (m, 2H); 1.90 (d, J=1.1 Hz, 3H); 1.81-1.74 (m, 1H);1.37-1.33 (m, 6H); 0.90 (s, 9H); 0.16 (d, J=21.5 Hz, 6H).

1.6. Preparation of Compound 7

To a solution of compound 6a (10.3 g, 21.08 mmol) in THF (50 mL) wasadded TBAF (1.0 M in THF, 25.3 mL, 25.3 mmol). The solution was stirredat rt for 1 h and concentrated to oil under reduced pressure. The crudeproduct was loaded onto a C18 column and eluted with 5 to 50% ACN inwater to afford compound 7. ¹H NMR (400 MHz, CDCl₃) δ=10.64 (s, 1H);7.24 (d, J=0.9 Hz, 1H); 6.98 (dt, J=21.9, 4.4 Hz, 1H); 6.12 (dt, J=18.8,1.6 Hz, 1H); 5.85 (s, 1H); 5.10-5.07 (m, 1H); 4.48-4.5 (d, J=4.8 Hz,1H); 4.16-4.07 (m, 4H); 2.34-2.29 (dd, J=13.2, 5.6 Hz, 1H); 1.87 (s,3H); 1.83-1.76 (m, 1H); 1.36-1.32 (m, 6H).

1.7. Preparation of Compound 8

To a solution of compound 7 (4.49 g, 18.97 mmol) and DIPEA (6.63 mL,37.9 mmol) in DCM (15 mL) at 0° C. was added 2-cyanoethylN,N-diisopropylchlorophosphoramidite (4.23 mL, 18.97 mmol). The mixturewas stirred at room temperature for 30 min, concentrated and loaded ontoa cyano column. The sample was eluted with 0 to 50% EtOAC in HEX withTEA (0.15%) to afford compound 8 as white foam. ¹H NMR (500 MHz, CDCl₃)δ=8.76 (s, 1H); 7.11 (t, J=1.0 Hz, 1H); 6.95-6.88 (m, 1H); 6.10-6.01 (m,1H); 5.96 (d, J=7.4 Hz, 1H); 4.95-4.92 (m, 1H); 4.60-4.57 (m, 1H);4.16-4.08 (m, 4H); 3.94-3.75 (m, 2H); 3.66-3.60 (m, 2H); 2.68-2.64 (m,2H); 2.40-2.24 (m, 1H); 1.91 (dd, J=2.7, 1.1 Hz, 3H); 1.36 (dt, J=7.2,1.6 Hz, 6H); 1.33-1.17 (m, 12H).

Example 2: Synthesis of Compound 12

Compound 12 is a phosphoramidite that can be used to generate single-and/or double-stranded siNA molecules comprising a “3daraT” or “3daraTs”nucleotide at the first nucleotide position of the molecule (position 1,which includes a 5′ cap; also referred to herein as “5-position 1”). Thechemical structure for 3daraT or 3daraTs can be found within Table 14,infra.

2.1. Preparation of Compound 9

To the solution of compound 4 (2.2 g, 3.34 mmol) in THF (10 ml) wascharged with 1.0 M TBAF in THF (4.01 ml, 4.01 mmol) at rt. The batch wasagitated for 1 h and then concentrated. The crude product was purifiedby silica with 0 to 95% EtOAC in Hex to afford compound 9 as colorlessoil. ¹H NMR (400 MHz, CD₃CN) δ=7.47-7.43 (m, 3H), 7.34-7.29 (m, 6H),7.26-7.22 (m, 1H), 6.89-6.85 (m, 4H), 5.70 (d, J=2.2 Hz, 1H), 4.48-4.43(m, 1H), 4.39-4.36 (m, 1H), 3.34-3.24 (m, 2H), 2.23-2.15 (m, 2H), 1.46(d, J=1.2 Hz, 3H)

2.2. Preparation of Compound 10

To the solution of compound 9 (455 mg, 0.835 mmol) in 2 ml DMF wascharged diphenyl carbonate (197 mg, 0.919 mmol) and sodium bicarbonate(2.11 mg, 0.025 mmol) at rt. It was agitated till all solid dissolve.The batch was heat at 100° C. for 1 hour and then quenched to mixture ofEtOAc and Brine. The aqueous was back extracted with EtOAC twice. Thecombined organic was concentrated to oil and purified by silica with 0to 100% acetone in Hex to afford compound 10 as white solid. ¹H NMR (400MHz, CD₃CN) δ=7.44-7.43 (m, 1H), 7.35-7.33 (m, 2H), 7.28-7.17 (m, 7H),6.82-6.79 (m, 4H), 6.08 (d, J=5.6 Hz, 1H), 5.40 (m, 1H), 4.60-4.54 (m,1H), 2.96-2.77 (m, 2H), 2.52-2.44 (m, 1H), 2.22-2.17 (m, 1H), 1.83 (d,J=1.2 Hz, 3H).

2.3. Preparation of Compound 11

To the compound 10 (410 mg, 0.779 mmol) in 1 ml ethanol was charged 1.0M NaOH solution (2.34 ml, 2.34 mmol). The mixture was agitated at rt for3 h, quenched with acetic acid (0.080 ml, 1.4 mmol). The crude productwas directly loaded on C18 column and purified with 0 to 75% MeCN inwater to afford compound 11 as white solid. ¹H NMR (400 MHz, CD₃CN)δ=9.27 (s, 1H) 7.48-7.45 (m, 3H), 7.34-7.29 (m, 6H), 7.26-7.21 (m, 1H),6.89-6.85 (m, 4H), 5.94 (d, J=4.7 Hz, 1H), 4.44 (dd, J=11.4, 5.0 Hz,1H), 4.21-4.15 (m, 1H), 3.33-3.25 (m, 2H), 2.27-2.30 (m, 2H), 1.65 (d,J=1.1 Hz, 3H)

2.4. Preparation of Compound 12

To the solution of compound 11 (379 mg, 0.696 mmol) and DIPEA (0.486 ml,2.78 mmol) in 2 ml DCM was charged 2-CyanoethylN,N-diisopropylchlorophosphoramidite (0.310 ml, 1.39 mmol). The mixturewas agitated at rt for 40 min and purified by cyano column with 0 to 85%EtOAC in Hex (0.15% Et₃N) to afford compound 12 as a white foam. ¹H NMR(500 MHz, CD₃CN) δ=9.00 (s, 1H) 7.48-7.45 (m, 2H), 7.37-7.29 (m, 7H),7.26-7.22 (m, 1H), 6.88-6.85 (m, 4H), 6.05, 6.02 (d, J=5.1 Hz, 1H),4.62-4.55 (m, 1H), 4.28-4.19 (m, 1H), 3.67-3.55 (m, 2H), 3.48-3.39 (m,2H), 3.35-3.26 (m, 2H), 2.59, 2.42 (m, 2H), 2.56-2.27 (m, 1H), 2.07-1.96(m, 1H), 1.66, 1.64 (d, J=1.1 Hz, 3H), 1.11-0.93 (m, 12H)

Example 3: Synthesis of Compound 14

Compound 14 is a phosphoramidite precursor that can be used to generatesingle- and/or double-stranded siNA molecules comprising a “3rT” or“3rTs” nucleotide at the first nucleotide position of the molecule(position 1, which includes a 5′ cap; also referred to herein as“5-position 1”). The chemical structure for 3rT or 3rTs can be foundwithin Table 14, infra. Generally, the same synthesis procedure can beused to make phosphoramidites that are used to generate single and/ordouble-stranded siNA molecules comprising 3rX or 3rXs at position one,wherein X is any heterocyclic base moiety.

3.1. Preparation of Compound 2a

To a solution of1-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione13 (4.00 g, 7.14 mmol) in anhydrous pyridine (30 ml) at rt was addedTBSCl (1.13 g, 7.49 mmol) and imidazole (1.21 g, 17.84 mmol). Theresulting mixture was stirred at rt for 18 h. Then, it was concentratedunder reduced pressure. EtOAc (50 ml), sat aq sodium citrate mono basic(20 ml) and water (10 ml) were added. Layers were separated and the aqlayer was extracted with EtOAc (5 ml×2). Combined organic solution waswashed with brine (5 ml), dried (MgSO₄), and concentrated. The crude wasdissolved in minimal amount of CH₂Cl₂ and loaded to silica column (120g, pre-equilibriated with Hex). It was eluted with 0 to 25% EtOAc in Hexto give 2′-OTBS product 2, and further elution with up to 40% EtOAc inHex provided 3′-OTBS product 2a. ¹H NMR (500 MHz, CDCl₃) δ 7.97 (s, 1H),7.59 (s, 1H), 7.40-7.25 (m, 9H), 6.84 (d, J=10 Hz, 4H), 5.97 (d, J=5 Hz,1H), 4.38 (m, 1H), 4.27 (m, 1H), 4.05 (m, 1H), 3.79 (s, 6H), 3.54 (d,J=5 Hz, 1H), 3.25 (d, J=10 Hz, 1H), 2.79 (d, J=10 Hz, 1H), 1.48 (s, 3H),0.86 (s, 9H), 0.06 (s, 3H), −0.03 (s, 3H).

3.2. Preparation of Compound 14

To a solution of 3′-OTBS-5′-ODMTr-5-methyluridine 2a (1.30 g, 1.93 mmol)in DCM (15 ml) at 0° C. was added tetrazole (54 mg, 0.771 mmol) and2-cyanoethyl N,N,N′,N′-tetraisopropylphosphoroamidite (0.795 ml, 2.50mmol). The reaction was warmed to rt and stirred for 60 h. Then, DCM (30ml) and sat aq NaHCO₃ (20 ml) were added. Layers were separated and theaq layer was extracted with DCM (2×10 ml). Combined organics were dried(MgSO₄), concentrated, and purified by column chromatography (elutedwith 0 to 35% EtOAc) to give amidite 14 as a solid. ¹H NMR (500 MHz,CDCl₃) δ 8.18 (s, 1H), 7.62 (s, 1H), 7.42-7.24 (m, 9H), 6.83 (d, J=10Hz, 4H), 6.17 (d, J=5 Hz, 1H), 4.28 (m, 2H), 4.09 (d, J=5 Hz, 1H), 3.79(m, 1H), 3.79 (s, 6H), 3.77 (m, 1H), 3.63 (m, 2H), 3.52 (d, J=10 Hz,1H), 3.26 (d, J=10 Hz, 1H), 2.60 (m, 2H), 1.59 (s, 3H), 1.16-1.14 (m,12H), 0.80 (s, 9H), 0.07 (s, 3H), −0.03 (s, 3H).

Example 4: Synthesis of Compound 16

Compound 16 is a phosphoramidite precursor that can be used to generatesingle- and/or double-stranded siNA molecules comprising a “3fluU” or“3fluUs” nucleotide at the first nucleotide position of the molecule(position 1, which includes a 5′ cap; also referred to herein as“5-position 1”). The chemical structure for 3fluU or 3fluUs can be foundwithin Table 13, infra. Generally, the same synthesis procedure can beused to make phosphoramidites that are used to generate single and/ordouble-stranded siNA molecules comprising 3fluX or 3fluXs at positionone, wherein X is any heterocyclic base moiety.

To a solution of1-((2R,3S,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione15 (0.78 g, 1.422 mmol) in anhydrous, degassed DCM (10 mL) at rt wasadded N,N-diisopropylethyl amine (0.551 g, 4.27 mmol) and 2-cyanoethylN,N-diisopropylchlorophosphoramidite (0.673 g, 2.84 mmol). The resultingmixture was stirred at rt for 30 min. Then, it was concentrated underreduced pressure. The crude product was dissolved in minimal amount ofCH₂Cl₂ and loaded to silica column (40 g, pre-equilibriated with Hex).It was eluted with 0 to 60% EtOAc in Hex to give amidite 16 as a whitesolid. ¹H NMR (400 MHz, CD₃CN) δ 1.08 (d, J=6.8 Hz, 3H); 1.19-1.18 (m,9H); 2.62 (dt, J=24.1, 6.0 Hz, 2H); 3.33 (dd, J=11.0, 3.1 Hz, 1H); 3.45(ddd, J=11.0, 7.3, 3.3 Hz, 1H); 3.63-3.62 (m, 2H); 3.74 (dt, J=8.1, 6.1Hz, 1H); 3.78 (s, 6H); 3.83-3.82 (m, 1H); 4.31-4.28, 4.38-4.35 (m, 1H);¹ 4.61-4.60 (m, 1H); 5.09-5.07, 5.22-5.20 (m, 1H); ¹ 5.41 (t, J=8.1 Hz,1H); 6.05 (t, J=6.3 Hz, 1H); 6.89-6.88 (m, 4H); 7.31-7.30 (m, 7H); 7.41(d, J=7.8 Hz, 2H); 7.55 (dd, J=8.2, 4.7 Hz, 1H); 9.02 (s, 1H). ³¹P NMR(162 MHz, CD₃CN) δ 151.8, 151.9.

Example 5: Oligonucleotide Synthesis: General Protocol

Oligonucleotide duplexes were prepared by individual synthesis of twocomplementary oligonucleotide sense and antisense strands. Thecomplementary strands of each duplex of a target sequence weresynthesized on solid support, such as on controlled pore glass. Aftercleavage of each strand from the solid support and deprotection of alloligonucleotide protecting groups, each strand was purifiedchromatographically with a reversed phase (C18) or anion exchange (SAX)resin. After purification, each sense strand was annealed with itscorresponding antisense strand and lyophilized to dryness.

The synthesis of each sense and antisense strand was accomplished on asolid support, such as controlled pore glass, using commerciallyavailable automated oligosynthesizers. The solid support was obtainedpre-loaded with the first (3′) nucleotide unit of the desired sequenceand placed in an appropriate column for the oligosynthesizer. The firstnucleotide was linked to the solid support via a succinate linkage andcontained a suitable acid sensitive protecting group (e.g., trityl,dimethoxytrityl) on the 5′-terminal hydroxyl group. The solid-phaseoligosynthesis employed synthetic procedures that are generally known inthe art. Elongation of the desired oligomeric sequence went through acycle of four steps: 1) acidic deprotection of the 5′-trityl protectinggroup; 2) coupling of the next nucleotide unit as the 5′-trityl (ordimethoxytrityl) protected phosphoramidite in the presence of anactivating agent, such as S-ethyl-tetrazole; 3) oxidation of the P(III)phosphite triester to the P(V) phosphate triester by an oxidizing agent,such as iodine; and, 4) capping any remaining unreacted alcohol groupsthrough esterification with an acylating agent, such as aceticanhydride. The phosphoramidites used were either derived from naturallyoccurring nucleotide units or from chemical modified versions of thesenucleotides. Typically, all phosphoramidites were prepared in solutionsof acetonitrile (or other suitable solvents or solvent mixtures, such asacetonitrile with some percentage of dimethyl formamide). The activatorfor phosphoramidite coupling was typically dissolved in acetonitrile. Anoxidizing agent, such as iodine, was dissolved in a suitable solventmixture, such as acetonitrile, pyridine and water. Acidic detritylationreagents, such as dichloroacetic acid or trichloroacetic acid, weredissolved in appropriate solvents, such as toluene or dichloromethane.Acylating capping reagents were, for example, a mixture of aceticanhydride, 2,6-lutidine and N-methyl-imidazole in acetonitrile. In placeof the oxidation of the phosphite triester to the phosphate triester,the P(III) intermediate may be converted to the phosphorothioate analogwith a sulfurizing reagent, such as phenyl-acetyl-disulfide, in asuitable solvent, such as a mixture of acetonitrile and pyridine. Inbetween each step of the oligonucleotide elongation, acetonitrile (oranother suitable solvent) was used to remove excess reagents and washthe solid support.

Oligonucleotide synthesis cycles were continued until the last (5′)nucleotide unit was installed onto the extended oligomer. After thefinal cycle, the 5′-trityl protecting group may or may not be removedfrom the oligonucleotide while it remains on the solid support. In someinstances, the 5′-terminal trityl was first removed by treatment with anacidic solution. After this deprotection, the solid support was treatedwith an appropriate base, such as aqueous methyl amine (at either roomtemperature or with mild heating) in order to cleave the oligonucleotidefrom the support, remove the cyanoethyl protecting groups on thephosphates and deprotect the acyl protecting groups on the nucleotidebases. Purification of these oligonucleotides would then be accomplishedby SAX chromatography. Typically, the oligonucleotide was eluted fromthe SAX resin with a gradient of an inorganic salt, such as sodiumchloride. Salt was removed from the purified samples by dialysis ortangential flow filtration. The desalted material was then lyophilizedor annealed directly with the corresponding complementary strand.Alternatively, the purified single strands were annealed prior toremoval of salt and then dialyzed after duplex formation.

In other instances, the 5′-trityl protecting group was left on theoligonucleotide during basic cleavage from the solid support anddeprotection of the oligonucleotide protecting groups. In this case, theoligonucleotide was purified with a reversed phase resin, such as C18.The presence of the trityl group allowed for the desired full lengtholigonucleotide to be retained on the resin, while undesired truncatedproducts that were acylated during the capping reaction were washed fromthe resin. These undesired oligonucleotides were removed from the resinwith a lower percentage of an organic solvent, such as acetonitrile.After this removal of failure products, the trityl group was removed byacidic treatment (aqueous trifluoroacetic acid) while still on the C18resin. After a salt exchange and extensive washing of the resin withwater, the desired deprotected product was eluted with a higherpercentage of organic solvent in water. These purified oligonucleotideswere then either lyophilized or annealed directly with the correspondingcomplementary strand.

If the oligonucleotide contained any ribose (2′-hydroxyl) nucleotides, amodified procedure was required to remove the silyl 2′-hydroxylprotecting groups. After cleaving the oligonucleotide from the solidsupport with aqueous base, the column was further washed with anappropriate solvent, such as DMSO, to remove any material remaining onthe column. After basic deprotection of the oligonucleotide, thereaction mixture then was treated with an appropriate fluoride reagent,such as triethylamine-hydrogen fluoride, to cleave all of the silylethers and expose the desired alcohols. After silyl deprotection wascomplete, an appropriate buffer was added to each sample in order toneutralize the solution prior to purification (either by SAX or C18).

In certain cases, a 5′-phosphonate-3′-phosphoramidite was coupled to the5′-terminus of an oligonucleotide. The oxidation reagent for thisincorporation was t-butyl hydroperoxide rather than iodine. When anoligonucleotide contained a 5′-phosphonate moiety, the methyl (or ethyl)phosphate esters were deprotected with an appropriate reagent, such asiodotrimethylsilane in pyridine/acetonitrile, while the oligonucleotidewas still on the solid support. After washing the support withacetonitrile, the oligonucleotide was treated with β-mercaptoethanol intriethylamine/acetonitrile. After further washing with acetonitrile, theoligonucleotide product was cleaved from the solid support and fullydeprotected with aqueous methylamine. The 5′-phosphonateoligonucleotides were purified by either SAX or C18 chromatography.

Each purified oligonucleotide was analyzed for purity by appropriatemethods, including reversed phase HPLC, SAX HPLC and capillary gelelectrophoresis. The identity of the oligonucleotide was confirmed bymass spectrometry, using an ionization technique such as ESI or MALDI.The yields of each oligonucleotide were assessed by UV (260 nm) with atheoretically derived extinction coefficient.

The corresponding sense and antisense strands were annealed by mixingequimolar amounts of each material. The appropriate amounts of eachstrand were approximated by UV (260 nm) measurements and theoreticalextinction coefficients. After the annealing process, the extent ofduplex formation and the presence of any excess single strand materialwere assessed by an appropriate chromatographic method, such as RP-HPLCor SAX. When appropriate, the sample was adjusted with additionalamounts of one of the two strands in order to completely anneal theremaining excess single strand. The final duplex material waslyophilized prior to delivery for further biochemical or biologicaltesting.

Example 6: Single-Stranded siNA Molecules Containing Modifications at5′-Position 1

Table 2 shows various chemically-modified single-stranded siNA moleculessynthesized using the protocol provided in Example 2 to 5. Thesingle-stranded siNA molecules within Table 2 are comprised of 21nucleotides (position 1 (5′) to position 21 (3′)) and containdifferential modifications at nucleotide position 1. The name of eachsiNA molecule is provided in column 1 and corresponds to the compositionof the nucleotide at position 1. An “siNA name” designated for an siNAmolecule in Table 2 is used in other Tables and Figures to representthat particular siNA molecule. Column 2 (“5′-position 1 nucleotide”)describes position 1 of each siNA molecule, comprising a nucleotide witha 5′ cap. The chemical structure of each of the 5′-position 1nucleotides is provided in Table 14, infra. For example, the 5′-position1 nucleotide of the benchmark (“BM”) siNA molecule, “p-omeU,” comprisesa chemically-modified uracil nucleotide with an O-methyl group at the 2′position of the sugar and a natural 5′ phosphate cap. The nucleotidesequence spanning positions 2-20 for each of the siNA molecules isdescribed in column 3 of Table 2, wherein the individual nucleotides areseparated by a semicolon. The chemical structure of each nucleotideindicated within column 3 is provided for in Table 15, infra. Forexample, the chemical structure of the nucleotide located at position 2of the BM siNA molecule, “flu,” is a chemically-modified uracilnucleotide with a fluoro group at the 2′ position of the sugar. Thesequence of nucleotide positions 2-20 of each of the siNA molecules ofTable 2 are the same (i.e., “(same)” with column 3). The 4^(th) columnof Table 2, “Nucleotide position 21-3′,” represents the 3′ mostnucleotide of the siNA molecules of Table 2, each represented by“omeUSup”. The structure of omeUSup is provided in Table 16, infra. TheSEQ ID NO: for each siNA molecule of Table 2 (nucleotide positions 1-21)is provided in column 5.

TABLE 2 Single-stranded siNA molecules that are 21 nucleotides in lengthand contain differential modifications at position 1. 5′- Nucleotide SEQposition 1 Nucleotide sequence - position ID siNA name nucleotideposition 2 to position 20 21-3′ NO. BM p-omeU fluU; omeU; fluC; omeUSup1 omeG; fluA; omeA; fluU; omeC; fluA; omeA; fluU; omeC; fluC; omeA;fluA; omeC; fluA; omeG; omeUs BMs p-omeUs (same) (same) 2 dT p-dT (same)(same) 3 dTs p-dTs (same) (same) 4 3dT p-3dT (same) (same) 5 3dTs p-3dTs(same) (same) 6 3dA p-3dA (same) (same) 7 3dAs p-3dAs (same) (same) 83dC p-3dC (same) (same) 9 3dCs p-3dCs (same) (same) 10 3dG p-3dG (same)(same) 11 3dGs p-3dGs (same) (same) 12 3priomeU p-3priomeU (same) (same)13 3priomeUs p-3priomeUs (same) (same) 14 3fluU p-3fluU (same) (same) 153fluUs p-3fluUs (same) (same) 16 3daraT p-3daraT (same) (same) 173daraTs p-3daraTs (same) (same) 18 3rT p-3rT (same) (same) 19 3rTsp-3rTs (same) (same) 20 vinylPmoeTs vinylPmoeTs (same) (same) 21vinylP3dT vinylP3dT (same) (same) 22 vinylP3dTs vinylP3dTs (same) (same)23

In Vitro Assay Targeting CTNNB1 (96-Well Plate Transfections)—

Cell Culture Preparation:

Mouse hepatoma cell line, Hepa1-6, was grown in Dulbecco's ModifiedEagle's Medium that was supplemented with 10% fetal bovine serum, 100μg/mL streptomycin, 100 U/mL penicillin, and 1% sodium pyruvate.

Transfection and Screening:

Cells were plated in all wells of tissue-culture treated, 96-well platesat a final count of 3500 cells/well in 100 μL, of the complete culturemedia. The cells were cultured overnight after plating at 37° C. in thepresence of 5% CO₂.

On the next day, complexes containing siNA and Lipofectamine™ RNAiMax(Invitrogen) were created as follows. A solution of RNAiMax diluted33-fold in OPTI-MEM was prepared. In parallel, solutions of the siNAsfor testing were prepared to a final concentration of 120 nM inOPTI-MEM. After incubation of RNAiMax/OPTI-MEM solution at roomtemperature for 5 min, an equal volume of the siNA solution and theRNAiMax solution were added together for each of the siNAs.

Mixing resulted in a solution of siNA/RNAiMax where the concentration ofsiNA was 60 nM. This solution was incubated at room temperature for 15minutes. After incubation, 20 μl of the solution was added to each ofthe relevant wells. The final concentration of siNA in each well was 10nM and the final volume of RNAiMax in each well was 0.45 μL.

For low concentration screens, siNAs were transfected at 1000, 100 or 10pM per well. For 12-point dose response curve studies, the siNA seriesare 6-fold serial dilution starting at 30 nM or 4-fold serial dilutionstarting at 40 nM. All transfections were set up as multiple biologicalreplicates.

The time of incubation with the RNAiMax-siRNA complexes was 24 hours andthere was no change in media between transfection and harvesting forscreening and dose response curve studies.

Cells-to-Ct and Reverse Transcription Reactions:

The culture medium was aspirated and discarded from the wells of theculture plates at the desired time points. The transfected cells werewashed once with 100 μL DPBS solution per well. Fifty microliters perwell of the Lysis Solution from the TaqMan® Gene Expression Cells-to-CT™Kit (Invitrogen, Cat#4399002) supplemented with DNase I was addeddirectly to the plates to lyse the cells. Five microliters per well ofStop Solution from the same kit was added to the plates 5 minutes later.The lysis plates were incubated for at least 2 minutes at roomtemperature. The plates can be stored for 2 hours at 4° C., or −80° C.for two months.

Each well of the reverse transcription plate required 10 μL, of 2×reverse transcriptase buffer, 1 μL, of 20× reverse transcription enzymeand 2 μL, of nuclease-free water. The reverse transcription master mixwas prepared by mixing 2× reverse transcription buffer, 20× reversetranscription enzyme mix, and nuclease-free water. 13 μL, of the reversetranscription master mix was dispensed into each well of the reversetranscription plate (semi-skirted). A separate reverse transcriptionplate was prepared for each cell plate. Seven microliters per lysatefrom the cell lysis procedure described above was added into each wellof the reverse transcription plate. The plate was sealed and spun on acentrifuge (1000 rpm for 30 seconds) to settle the contents to thebottom of the reverse transcription plate. The plate was placed in athermocycler at 37° C. for 60 min, 95° C. for 5 min, and 4° C. until theplate is removed from the thermocycler. Upon removal, if not usedimmediately, the plate was frozen at −20° C.

Quantitative RT-PCR (Taqman):

A series of probes and primers were used to detect the various mRNAtranscripts of the genes of CTNNB1 and GAPDH. All Taqman probes andprimers for the experiments here-in described were supplied aspre-validated sets by Applied Biosystems, Inc. (see Table 3).

TABLE 3 Probes and primers used to carry out Real-Time RT/PCR (Taqman)reactions for CTNNB1 mRNA analysis. Species Gene ABI Cat.# Human CTNNB1Hs00355045_m1 Human GAPDH 4310884E Mouse CTNNB1 Mm00483033_m1 MouseGAPDH 4352339E

The assays were performed on an ABI 7900HT instrument, according to themanufacturer's instructions. A TaqMan Gene Expression Master Mix(provided in the Cells-toCT™ Kit, Invitrogen, Cat #4399002) was used.The PCR reactions were carried out at 50° C. for 2 min, 95° C. for 20sec followed by 40 cycles at 95° C. for 1 min and 60° C. for 20 sec.

Within each experiment, the baseline was set in the exponential phase ofthe amplification curve, and based on the intersection point of thebaselines with the amplification curve; a Ct value was assigned by theinstrument.

Calculations:

The expression level of the gene of interest and % inhibition of geneexpression (% KD) was calculated using Comparative Ct method:ΔCt=Ct _(Target) −Ct _(GAPDH)ΔΔCt(log 2(fold change))=ΔCt _((Target siNA)) −ΔCt _((NTC))Relative expression level=2^(−ΔΔCt)% KD=100×(1−2^(−ΔΔCt))

The non-targeting control siNA was, unless otherwise indicated, chosenas the value against which to calculate the percent inhibition(knock-down) of gene expression, because it is the most relevantcontrol.

Additionally, only normalized data, which reflects the general health ofthe cell and quality of the RNA extraction, was examined. This was doneby looking at the level of two different mRNAs in the treated cells, thefirst being the target mRNA and the second being the normalizer mRNA.This allowed for elimination of siNAs that might be potentially toxic tocells rather than solely knocking down the gene of interest. This wasdone by comparing the Ct for GAPDH in each well relative to the GAPDH Ctfor the entire plate.

All calculations of IC50s were performed using R.2.9.2 software. Thedata were analyzed using the sigmoidal dose-response (variable slope)equation for simple ligand binding. In all of the calculations of thepercent inhibition (knock-down), the calculation was made relative tothe normalized level of expression of the gene of interest in thesamples treated with the non-targeting control (Ctrl siRNA) unlessotherwise indicated.

Results—

Subsets of the single-stranded siNA molecules of Table 2 were screenedin Hepa1-6 cells transfected with RNAiMax. Table 4 and FIG. 1A summarizethe knock-down activities of regular 3′-5′ linked single-stranded siNAmolecules such as BM (benchmark), BMs, dT, and dTs, and new 2′-5 linkedsingle-stranded siNA molecules such as 3dX and 3dXs, where X is T, A, Cor G. The “s” designation within the siNA name indicates aphosphorothioate internucleotide linkage between nucleotide position 1and 2 of the molecule. The “d” designation within the siNA nameindicates a deoxy modification at nucleotide position 1 of the molecule(e.g., dT siNA has a deoxy modification at the 2^(nd) carbon position ofthe sugar at nucleotide position 1 and a 3′-5′ phosphodiesterinternucleotide linkage between nucleotide positions 1 and 2; 3dT siNAhas 2 H at the 3^(rd) carbon position of the sugar at nucleotideposition 1 and a 2′-5′ phosphodiester internucleotide linkage betweennucleotide positions 1 and 2).

Each of the 2′-5′ linked 3dX and 3dXs siNA molecules showed knock-downin this assay. In particular, the 2′-5′ linked 3dT and 3dTs siNAmolecules showed higher knock-down activities than the 3′-5′ linked BM,BMs, dT, and dTs at all four concentrations (100 nM, 10 nM, 1 nM, and0.1 nM). Among 2′-5′ linked single-stranded siNA molecules, 3dT and 3dTsshowed higher knock down activities than other 3dX and 3dXs, where X wasA, C or G at all four concentrations (100 nM, 10 nM, 1 nM, and 0.1 nM).The 3dA, 3dAs, 3dC and 3dCs siNA molecules displayed knock-down activitythat was slightly better or the same as benchmark molecules.

TABLE 4 In vitro transfection-based knock down activity for a subset ofthe single-stranded siNA molecules provided in Table 2 (see also FIG.1A). siNA ddCT ddCT ddCT ddCT name (100 nM) (10 nM) (1 nM) (0.1 nM) BM1.324 0.796 0.164 −0.115 BMs 1.073 1.474 0.246 0.005 dT 2.553 2.8540.996 0.350 dTs 1.543 2.159 0.636 0.175 3dT 3.168 3.474 2.316 1.45 3dTs2.593 3.519 2.291 0.92 3dA 1.667 1.074 0.139 −0.008 3dAs 1.484 1.3310.249 0.040 3dC 1.462 1.421 0.262 0.118 3dCs 1.239 1.494 0.269 0.115 3dG0.614 0.191 −0.043 −0.003 3dGs 0.459 0.229 0.004 −0.013

Table 5 and FIG. 1B summarize the knock-down activities for anothersubset of the siNA molecules shown in Table 2, comparing the activitiesof 2′-5′ linked single-stranded siNA molecules with various structuralchanges in the nucleotide at position 1. Each of the 2′-5′ linkedsingle-stranded siNA molecules displayed knock-down activity in theassay. In particular, 3dT and 3dTs siNA molecules showed higher knockdown activities than 3priomeU, 3priomeUs, 3fluU, 3fluUs, 3daraT,3daraTs, 3rT, and 3rTs at all four concentrations (100 nM, 10 nM, 1 nM,and 0.1 nM).

TABLE 5 In vitro transfection-based knock down activity for a subset ofthe single-stranded siNA molecules provided in Table 2 (see also FIG.1B). siNA ddCT ddCT ddCT ddCT name (100 nM) (10 nM) (1 nM) (0.1 nM) BM1.324 0.796 0.164 −0.115 BMs 1.073 1.474 0.246 0.005 3dT 3.168 3.4742.316 1.45 3dTs 2.593 3.519 2.291 0.92 3priomeU 0.768 0.514 0.051 0.0853priomeUs 0.448 0.579 −0.024 −0.090 3fluU 0.921 0.825 0.021 −0.3333fluUs 0.481 0.373 0.126 0.155 3daraT −0.046 0.669 0.070 0.289 3daraTs0.249 0.074 0.040 −0.001 3rT 2.188 0.919 0.135 −0.245 3rTs 0.908 0.614−0.095 −0.350

Subsets of the single-stranded siNA molecules of Table 2 were assayedfurther for 12-point dose response curve. Table 6 summarizes the knockdown activity of the single-stranded siNA molecules. 3dT and 3dTs showedlower IC50 values (column 2) and higher maximum knock-down activity(column 3) than other single-stranded siNA molecules, such as BM, BMs,dT, dTs, vinylPmoeTs, vinylP3dT, and vinylP3dTs.

TABLE 6 Dose-response in vitro transfection-based knock down activityfor a subset of the single-stranded siNA molecules provided in Table 2.Max KD siNA name IC50 (nM) (ddCT) BM 2.189 −2.428 BMs 4.250 −1.899 dT0.813 −2.779 dTs 0.848 −2.167 3dT 0.355 −2.915 3dTs 0.580 −2.834vinylPmoeTs 5.831 −1.582 vinylP3dT 3.341 −1.994 vinylP3dTs 4.196 −1.950

Example 7: Single-Stranded siNA Molecules Containing Internal 3dXNucleotide Modifications

The single-stranded siNA molecules within Table 7 are comprised of 21nucleotides (position 1 (5′) to position 21 (3′)), and each moleculecontains a single 3dX nucleotide, wherein X is base A, T, C, or G,located at different nucleotide positions. The name of each siNAmolecule is provided in column 1 and corresponds to the nucleotideposition containing the 3dX nucleotide and the base used at thatposition (e.g., “2(T)” indicates a 3dT nucleotide at position 2). An“siNA name” designated for an siNA molecule in Table 7 is used in otherTables and Figures to represent that particular siNA molecule. Column 2of Table 7, “5′-position 1 nucleotide”, represents the 5′ mostnucleotide of the siNA molecules of Table 7, each represented by“p-omeU” (see Table 14, infra, for the p-omeU structure). The nucleotidesequence spanning positions 2-20 for each of the siNA molecules isdescribed in column 3 of Table 7, wherein the individual nucleotides areseparated by a semicolon. The chemical structure of each nucleotideindicated within column 3 is provided for in Table 15, infra. The 3dXnucleotides within column 3 are underlined. The 4^(th) column of Table7, “Nucleotide position 21-3′”, represents the 3′ most nucleotide of thesiNA molecules of Table 7, each represented by “omeUSup” (see Table 16,infra, for the omeUSup structure). The SEQ ID NO: for each siNA moleculeof Table 7 (positions 1-21) is provided in column 5.

TABLE 7 Single-stranded siNA molecules containing a single, internal 3dXnucleotide (underlined in column 3). 5′- Nucleotide siNA position 1position 21- SEQ ID name nucleotide Nucleotide sequence - position 2 toposition 20 3′ NO.  2(T) p-omeU 3dT; omeU; fluC; omeG; fluA; omeA; fluU;omeC; fluA; omeA; omeUSup 24 fluU; omeC; fluC; omeA; fluA; omeC; fluA;omeG; omeUs  3(T) (same) fluU; 3dT; fluC; omeG; fluA; omeA; fluU; omeC;fluA; omeA; (same) 25 fluU; omeC; fluC; omeA; fluA; omeC; fluA; omeG;omeUs  4(C) (same) fluU; omeU; 3dC; omeG; fluA; omeA; fluU; omeC; fluA;omeA; (same) 26 fluU; omeC; fluC; omeA; fluA; omeC; fluA; omeG; omeUs 5(G) (same) fluU; omeU; fluC; 3dG; fluA; omeA; fluU; omeC; fluA; omeA;(same) 27 fluU; omeC; fluC; omeA; fluA; omeC; fluA; omeG; omeUs  6(A)(same) fluU; omeU; fluC; omeG; 3dA; omeA; fluU; omeC; fluA; omeA; (same)28 fluU; omeC; fluC; omeA; fluA; omeC; fluA; omeG; omeUs  8(T) (same)fluU; omeU; fluC; omeG; fluA; omeA; 3dT; omeC; fluA; omeA; (same) 29fluU; omeC; fluC; omeA; fluA; omeC; fluA; omeG; omeUs  9(C) (same) fluU;omeU; fluC; omeG; fluA; omeA; fluU; 3dC; fluA; omeA; (same) 30 fluU;omeC; fluC; omeA; fluA; omeC; fluA; omeG; omeUs 10(A) (same) fluU; omeU;fluC; omeG; fluA; omeA; fluU; omeC; 3dA; omeA; (same) 31 fluU; omeC;fluC; omeA; fluA; omeC; fluA; omeG; omeUs 12(T) (same) fluU; omeU; fluC;omeG; fluA; omeA; fluU; omeC; fluA; omeA; (same) 32 3dT; omeC; fluC;omeA; fluA; omeC; fluA; omeG; omeUs 13(C) (same) fluU; omeU; fluC; omeG;fluA; omeA; fluU; omeC; fluA; omeA; (same) 33 fluU; 3dC; fluC; omeA;fluA; omeC; fluA; omeG; omeUs 14(C) (same) fluU; omeU; fluC; omeG; fluA;omeA; fluU; omeC; fluA; omeA; (same) 34 fluU; omeC; 3dC; omeA; fluA;omeC; fluA; omeG; omeUs 15(A) (same) fluU; omeU; fluC; omeG; fluA; omeA;fluU; omeC; fluA; omeA; (same) 35 fluU; omeC; fluC; 3dA; fluA; omeC;fluA; omeG; omeUs 16(A) (same) fluU; omeU; fluC; omeG; fluA; omeA; fluU;omeC; fluA; omeA; (same) 36 fluU; omeC; fluC; omeA; 3dA; omeC; fluA;omeG; omeUs 17(C) (same) fluU; omeU; fluC; omeG; fluA; omeA; fluU; omeC;fluA; omeA; (same) 37 fluU; omeC; fluC; omeA; fluA; 3dC; fluA; omeG;omeUs 18(A) (same) fluU; omeU; fluC; omeG; fluA; omeA; fluU; omeC; fluA;omeA; (same) 38 fluU; omeC; fluC; omeA; fluA; omeC; 3dA; omeG; omeUs19(G) (same) fluU; omeU; fluC; omeG; fluA; omeA; fluU; omeC; fluA; omeA;(same) 39 fluU; omeC; fluC; omeA; fluA; omeC; fluA; 3dG; omeUs 20(T)(same) fluU; omeU; fluC; omeG; fluA; omeA; fluU; omeC; fluA; omeA;(same) 40 fluU; omeC; fluC; omeA; fluA; omeC; fluA; omeG; 3dTs

Results—

The single-stranded siNA molecules of Table 7 were screened in Hepa1-6cells transfected with RNAiMax, as in Example 6. Table 8 and FIG. 2Asummarize the knock-down activities of these single-stranded siNAmolecules containing internal 3dX nucleotide modifications. Each of thesingle-stranded siNA molecules tested displayed knock-down activity inthe assay. In particular, the 3dT siNA molecule showed higher knock-downactivity than the other single-stranded siNA molecules with internal3dX, wherein X was base A, T, C, or G, at all four concentrations (100nM, 10 nM, 1 nM, and 0.1 nM).

TABLE 8 In vitro transfection-based knock down activity for each of thesingle-stranded siNA molecules provided in Table 7, and the BM and 3dTsiNA molecules (see Table 2) (see also FIG. 2A). siNA ddCT ddCT ddCTddCT name (100 nM) (10 nM) (1 nM) (0.1 nM) BM 1.324 0.796 0.164 −0.1153dT 3.168 3.474 2.316 1.45  2 (T) 0.107 −0.194 −0.043 −0.080  3 (T)0.854 0.199 −0.163 −0.175  4 (C) 0.097 0.141 0.114 0.077  5 (G) 0.2590.234 0.044 0.048  6 (A) 0.797 0.139 −0.086 −0.043  8 (T) 0.604 0.3160.039 −0.035  9 (C) 0.122 0.164 0.039 0.055 10 (A) 0.539 0.526 −0.026−0.133 12 (T) 0.892 0.564 0.122 −0.042 13 (C) 0.134 0.084 0.064 −0.05014 (C) 0.184 0.174 0.099 0.062 15 (A) 0.317 0.201 0.004 −0.042 16 (A)0.562 0.259 −0.053 −0.025 17 (C) 0.752 1.079 0.089 0.005 18 (A) 0.8370.519 0.022 0.025 19 (G) 1.199 0.826 0.257 0.107 20 (T) 1.177 0.9510.209 0.043

Example 8: Single-Stranded siNA Molecules Containing a 3dT-RelatedNucleotide at Position 1 and Multiple Phosphorothioate InternucleotideLinkages

Table 9 shows various chemically-modified single-stranded siNA moleculessynthesized using the protocol provided in Example 5. Thesingle-stranded siNA molecules within Table 9 are comprised of 21nucleotides (position 1 (5′) to position 21 (3′)), each containing asingle 3dT or 3dTs nucleotide at position 1 and multiplephosphorothioate internucleotide linkages. A 3dTs nucleotide contains aphosphorothioate internucleotide linking group. The name of each siNAmolecule is provided in column 1 and corresponds to the number ofphosphorothioate internucleotide linkages contained within an siNAmolecule (e.g., the “14S” siNA molecule contains 14 phosphorothioateinternucleotide linkages). An “siNA name” designated for an siNAmolecule in Table 9 is used in other Tables and Figures to representthat particular siNA molecule. Column 2 of Table 9, “5′-position 1nucleotide”, represents the 5′ most nucleotide of the siNA molecules ofTable 9, each represented by either “p-3dT” or “p-3dTs” (for structures,see Table 14, infra). The nucleotide sequence spanning positions 2-20for each of the siNA molecules is described in column 3 of Table 9,wherein the individual nucleotides are separated by a semicolon. Thechemical structure of each nucleotide indicated within column 3 isprovided for in Table 15, infra. The phosphorothioate internucleotidelinking groups are indicated with an “s”, and the nucleotide positionsof the phosphorothioate internucleotide linkages are indicated in column4. The 5th column of Table 9, “Nucleotide position 21-3′”, representsthe 3′ most nucleotide of the siNA molecules of Table 9, eachrepresented by “omeUSup” (for structure, see Table 16, infra). The SEQID NO: for each siNA molecule of Table 9 (positions 1-21) is provided incolumn 6.

TABLE 9 Single-stranded siNA molecules containing a 3dT or 3dTsnucleotide at position 1 and multiple phosphorothioate internucleotidelinkages (“S-linkage”). 5′- Nucleotide S-linkage SEQ siNA position 1Nucleotide sequence - position 21- nucleotide ID name nucleotideposition 2 to position 20 3′ positions NO. 14S p-3dTs fluUs; omeU;fluCs; omeG; fluAs; omeA; fluUs; omeUSup 1, 2, 4, 6, 8, 10, 41 omeC;fluAs; omeA; fluUs; omeC; fluCs; omeAs; 12, 14-20 fluAs; omeCs; fluAs;omeGs; omeUs 13S p-3dTs fluU; omeU; fluCs; omeG; fluAs; omeA; fluUs;(same) 1, 4, 6, 8, 10, 12, 42 omeC; fluAs; omeA; fluUs; omeC; fluCs;omeAs; 14-20 fluAs; omeCs; fluAs; omeGs; omeUs 12S p-3dT fluU; omeU;fluCs; omeG; fluAs; omeA; fluUs; (same) 4, 6, 8, 10, 12, 43 omeC; fluAs;omeA; fluUs; omeC; fluCs; omeAs; 14-20 fluAs; omeCs; fluAs; omeGs; omeUs10S p-3dT fluU; omeU; fluC; omeG; fluA; omeA; fluU; omeC; (same) 11-2044 fluA; omeAs; fluUs; omeCs; fluCs; omeAs; fluAs; omeCs; fluAs; omeGs;omeUs  8S p-3dT fluU; omeU; fluC; omeG; fluA; omeA; fluU; omeC; (same)13-20 45 fluA; omeA; fluU; omeCs; fluCs; omeAs; fluAs; omeCs; fluAs;omeGs; omeUs  6S p-3dT fluU; omeU; fluC; omeG; fluA; omeA; fluU; omeC;(same) 15-20 46 fluA; omeA; fluU; omeC; fluC; omeAs; fluAs; omeCs;fluAs; omeGs; omeUs  4S p-3dT fluU; omeU; fluC; omeG; fluA; omeA; fluU;omeC; (same) 17-20 47 fluA; omeA; fluU; omeC; fluC; omeA; fluA; omeCs;fluAs; omeGs; omeUs

Results—

The single-stranded siNA molecules of Table 9 were screened in Hepa1-6cells transfected with RNAiMax, as in Example 6. Table 10 and FIG. 2Bsummarize the knock-down activities of these single-stranded siNAmolecules containing multiple phosphorothioate incorporations and a2′-5′ linkage at position 1. Each of the single-stranded siNA moleculesdisplayed knock-down activity in the assay that was equal to or betterthan that of the benchmark siNA molecules (BM, BMs). In particular, the3dT and 3dTs siNA molecules showed higher knock down activities than anyother single-stranded siNA molecules with multiple phosphorothioateincorporations at all four concentrations (100 nM, 10 nM, 1 nM, and 0.1nM).

TABLE 10 In vitro transfection-based knock down activity for each of thesingle-stranded siNA molecules provided in Table 8, and BM, BMs, 3dT and3dTs siNA molecules (see also FIG. 2B). siNA ddCT ddCT ddCT ddCT name(100 nM) (10 nM) (1 nM) (0.1 nM) BM 1.324 0.796 0.164 −0.115 BMs 1.0731.474 0.246 0.005 3dT 3.168 3.474 2.316 1.45 3dTs 2.593 3.519 2.291 0.9214S 1.982 2.376 0.717 0.095 13S 1.479 1.114 −0.048 −0.040 12S 1.6421.336 −0.016 −0.055 10S 2.729 2.609 0.309 0.115  8S 2.477 2.391 0.579−0.017  6S 2.274 2.449 0.754 −0.022  4S 2.504 2.216 0.794 −0.023

Example 9: Double-Stranded siNA Molecules Containing Modifications at5′-Position 1

Table 11 shows various chemically-modified double-stranded siNAmolecules synthesized using the protocol provided in Examples 1 and 5.The double-stranded siNA molecules within Table 11 contain a sensestrand (also known as the passenger strand) and an antisense strand(also known as the guide strand), wherein each strand is comprised of 21nucleotides (position 1 (5′) to position 21 (3′)) and containsdifferential modifications at position 1. The name of each siNA moleculeis provided in column 1 and corresponds to the composition of thenucleotide at position 1 of the sense and/or antisense strand of theduplex. Column 2 of Table 11, “Strand”, indicates whether the particularsequence is the sense (S) or antisense (A/S) strand of the duplex.Column 3 of Table 11, “5-position 1 nucleotide”, describes position 1 ofthe sense and antisense strands of the double-stranded siNA molecules ofTable 11, each comprising of a nucleotide with a 5′ cap. The chemicalstructure of each of the 5′-position 1 nucleotides is provided in Table14, infra. The nucleotide sequence spanning positions 2-20 for each ofthe sense and antisense strands of the duplex siNA molecules isdescribed in column 4 of Table 11, wherein the individual nucleotidesare separated by a semicolon. The chemical structure of each nucleotideindicated within column 4 is provided for in Table 15, infra. The 5thcolumn of Table 11, “Nucleotide position 21-3′”, represents the 3′ mostnucleotide of the sense or antisense strand of the double-stranded siNAmolecules, each represented by “omeU-iBSup” or “omeUSup” (forstructures, see Table 16, infra). The SEQ ID NO: for each strand of thedouble-stranded siNA molecules of Table 11 (positions 1-21) is providedin column 6. Each siNA molecule in Table 11 has 3′ overhangs at bothends of the molecule.

TABLE 11 Double-stranded siNA molecules, each containing a sense (S) andan antisense (A/S) strand that are 21 nucleotides in length and containdifferential modifications at position 1. 5′- Nucleotide SEQ siNAposition 1 position 21- ID name Strand nucleotide Nucleotide sequence -position 2 to position 20 3′ NO: ds TGN S tetraGalNAcLys- omeU; fluG;omeU; omeU; fluG; fluG; fluA; omeU; omeU- 49 BMs 6amiL-iB- omeU; fluG;fluA; omeU; omeU; omeC; fluG; fluA; iBSup omeC fluA; fluA; omeUs; A/SomeUs fluUs; omeUs; fluC; omeG; fluA; omeA; fluU; omeC; omeUSup 48 fluA;omeA; fluU; omeC; fluC; omeA; fluA; omeC; fluA; omeG; omeUs ds BMs SiB-omeC (same as ds TGN BMs S strand) omeU- 50 iBSup A/S p-omeUs (sameas ds TGN BMs A/S strand) omeUSup 51 ds 3dT S iB-omeC (same as ds TGNBMs S strand) omeU- 50 iBSup A/S p-3dT (same as ds TGN BMs A/S strand)omeUSup 52 ds 3dTs S iB-omeC (same as ds TGN BMs S strand) omeU- 50iBSup A/S p-3dTs (same as ds TGN BMs A/S strand) omeUSup 53 ds dT SiB-omeC (same as ds TGN BMs S strand) omeU- 50 iBSup A/S p-dT (same asds TGN BMs A/S strand) omeUSup 54 ds dTs S iB-omeC (same as ds TGN BMs Sstrand) omeU- 50 iBSup A/S p-dTs (same as ds TGN BMs A/S strand) omeUSup55 ds TGN S tetraGalNAcLys- (same as ds TGN BMs S strand) omeU- 49vinylP- 6amiL-iB- iBSup omeU omeC A/S vinylP- (same as ds TGN BMs A/Sstrand) omeUSup 56 omeU ds TGN S tetraGalNAcLys- (same as ds TGN BMs Sstrand) omeU- 49 vinylP- 6amiL-iB- iBSup omeUs omeC A/S vinylP- (same asds TGN BMs A/S strand) omeUSup 57 omeUs ds TGN S tetraGalNAcLys- (sameas ds TGN BMs S strand) omeU- 49 vinylP- 6amiL-iB- iBSup 3dT omeC A/SvinylP3dT (same as ds TGN BMs A/S strand) omeUSup 58 ds TGN StetraGalNAcLys- (same as ds TGN BMs S strand) omeU- 49 vinylP- 6amiL-iB-iBSup 3dTs omeC A/S vinylP- (same as ds TGN BMs A/S strand) omeUSup 593dTs ds TGN S tetraGalNAcLys- (same as ds TGN BMs S strand) omeU- 49vinylP- 6amiL-iB- iBSup moeT omeC A/S vinylP- (same as ds TGN BMs A/Sstrand) omeUSup 60 moeT

Results—

The double-stranded siNA molecules of Table 11 were assayed in vitro for12-point dose response curve as described in Example 6. Table 12summarizes the knock-down activity of the double-stranded siNAmolecules. New siNA molecules with 2′-5′ linked nucleotides at position1 of the antisense strand, such as ds 3dTs, ds TGN vinylP3dT, and ds TGNvinylP3dTs showed sub pico molar knock-down activity (IC50), which wasat least 10 times more potent than siNA molecules with regular 3′-5′linked nucleotides at position 1 of the antisense strand, such as ds TGNBMs, ds BMs, and ds dTs. This high knock-down activity of the siNAmolecules with 2′-5′ linked nucleotides at position 1 of the antisensestrand was even comparable to regular 3′-5′ linked siNA molecules(position 1 of antisense strand) with non-hydrolysable phosphonic acidat 5′-end of antisense strand, such as ds TGN vinylPomeU and ds TGNvinylPomeUs,

TABLE 12 In vitro transfection-based knock down activity for a subset ofthe double-stranded siNA molecules provided in Table 11. IC50 Max KDsiNA name (pM) (ddCT) ds TGN BMs 62 −2.96 ds BMs 5.63 −3.57 ds 3dT 0.68−3.48 ds 3dTs 0.17 −3.51 ds dTs 5.38 −3.66 ds TGN vinylPomeU 0.18 −3.57ds TGN vinylPomeUs 0.14 −3.49 ds TGN vinylP3dT 0.33 −3.49 ds TGNvinylP3dTs 0.36 −3.68

Example 10: In Vivo Studies (Mice) with Double-Stranded siNA MoleculesContaining Modifications at 5′-Position 1

Mice were dosed via subcutaneous injection with PBS control orGalNAc-conjugated siNAs at 5 mg/kg or 1 mg/kg. The animals weresacrificed 72 hrs after the dosing. Liver punches were collected for RNApurification. Total RNA was purified using RNeasy 96 kit (Qiagen,Cat#74182). cDNA is generated from total RNA using High Capacity cDNAReverse Transcription Kit (Invitrogen Cat#: 4368813). Quantitative PCRreactions are performed with TaqMan Universal PCR Master Mix (Cat#:4304437). Mouse CTNNBI TaqMan Gene Expression Assay (Mm00483033_ml) andmouse GAPDH TaqMan Gene Expression Assay were used to monitor the mRNAlevels of both transcripts in liver tissue. The expression level ofCTNNB 1 was normalized against GAPDH.

Results—

Subsets of the double-stranded siNA molecules of Table 11 were assayedin vivo (mice). The sense strands of these siNA molecules were linked totetraGalNAc Lys which targets the asialoglycoprotein receptor expressedon the surface of hepatoma cell lines. Table 13 summarizes theknock-down activities of the siNA molecules. ds TGN BMs which was notlinked to non-hydrolysable phosphorus showed low activity in both 5mg/kg and 1 mg/kg dose regimen. Among the siNA molecules which werelinked to non-hydrolysable phosphorus, both the siNA molecules withregular 3′-5′ linked oligonucleotides at position 1, such as ds TGNvinylPmoeT, ds TGN vinylPomeU, and ds TGN vinylPomeUs, and the siNAmolecules with new 2′-5′ linked oligonucleotides, such as ds TGNvinylP3dT and ds TGN vinylP3dTs, showed comparably high knock downactivity in both 5 mg/kg and 1 mg/kg dose regimen.

TABLE 13 In vivo mouse knock down activity (subcutaneous dose) for asubset of the double-stranded siNA molecules provided in Table 11. % KDsiNA name at 5 mg/kg at 1 mg/kg ds TGN BMs 43.65 19.51 ds TGN vinylPmoeT62.97 27.35 ds TGN vinylP3dT 62.64 22.04 ds TGN vinylP3dTs 64.21 21.03ds TGN vinylPomeU 69.43 14.80 ds TGN vinylPomeUs 70.64 38.12

Example 11

Chemical structures of the chemically-modified nucleotides used togenerate the single- and double-stranded siNA molecules exemplified inExamples 6-10.

TABLE 14 Structure of 5′-position 1 nucleotides contained within thesingle- or double-stranded siNA molecules exemplified in Tables 2, 7, 9and/or 11. 5′- position 1 nucleotide Structure p-omeU

p-omeUs

p-3dX B = Base T, A, C, G

p-3dXs B = Base T, A, C, G

p-dT

p-dTs

p-3priomeU

p-3priomeUs

p-3fluU

p-3fluUs

p-3daraT

p-3daraTs

p-3rT

p-3rTs

vinylP3dT

vinylP3dTs

vinylPmoeT

vinylPmoeTs

vinylPomeU

vinylPomeUs

iB-omeC

omeUs

tetraGalNAc Lys-6amiL- iB-omeC

TABLE 15 Structure of internally-located nucleotides (i.e., positions2-20) contained within the single- or double-stranded siNA moleculesexemplified in Tables 2, 7, 9 and/or 11. Internal nucleotide (positions2-20) Structure omeX X = B = Base U, G, C, A

omeXs X = B = Base U, G, C, A

3dX X = B = Base T, G, C, A

3dXs X = B = Base T, G, C, A

fluX X = B = Base U, G, C, A

fluXs X = B = Base U, G, C, A

TABLE 16 Structure of nucleotide position 21-3′ nucleotides exemplifiedin Tables 2, 7, 9 and/or 11. Nucleotide position 21-3′ Structure omeUsup

omeU-iBSup

What is claimed is:
 1. A short interfering nucleic acid (siNA) moleculecapable of mediating RNA interference comprising an antisense strandthat is complementary to a nucleic acid target, wherein said antisensestrand comprises a 5′ modified nucleotide having Formula II:

wherein: A is —OC(R³)₂—, —C(R³)₂O—, —C(R³)₂—, —C(R³)₂C(R³)₂— or—CR³═CR³—; B is any heterocyclic base moiety; D¹ and D^(1′) areindependently selected from hydroxyl, —OR⁴, —SR⁴, or —N(R⁴)₂; E is O, S,—NR⁵, —N—N(R⁴)₂ or —N—OR⁴; J is an internucleoside linking group linkingthe 5′ modified nucleotide of Formula II to the sugar moiety of anadjacent nucleotide of the siNA molecule; R¹ and R^(1′) areindependently selected from H, hydroxyl, halogen, C₁₋₆ alkyl, —OR⁶,—N(R⁶)₂, or together form ═O or ═CH₂; R² is H, C₁₋₆ alkyl or C₂₋₆alkenyl; R³ and R⁵ are independently selected from H, hydroxyl, halogen,C₁₋₁₀ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, aryl,

wherein R′ is selected from H or C₁₋₄ alkyl (which is optionallysubstituted with one to three substituents independently selected from—SR¹¹, aryl, heteroaryl, amino, hydroxyl, oxo or —NH—C═(NH)NH₂, whereinthe aryl and heteroaryl are optionally substituted with hydroxyl), andR″ is selected from H, C₁₋₁₈ alkyl or aryl; R⁴ is independently selectedfrom H, C₁₋₁₀ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, aryl,

wherein R′ is selected from H or C₁₋₄ alkyl (which is optionallysubstituted with one to three substituents independently selected from—SR¹¹, aryl, heteroaryl, amino, hydroxyl, oxo or —NH—C═(NH)NH₂, whereinthe aryl and heteroaryl are optionally substituted with hydroxyl), andR″ is selected from H, C₁₋₁₈ alkyl or aryl; R⁶ is independently selectedfrom H, C₁₋₆ alkyl (which is optionally substituted with —OR⁷, —SR⁷,—N(R⁸)₂, or (═O)—NR⁹ or from one to three halogen), C₂₋₆ alkenyl, C₂₋₆alkynyl, aryl,

wherein R′ is selected from H or C₁₋₄ alkyl (which is optionallysubstituted with one to three substituents independently selected from—SR¹⁰, aryl, heteroaryl, amino, hydroxyl, oxo, —NH—C═(NH)NH₂, whereinthe aryl and heteroaryl are optionally substituted with hydroxyl), andR″ is selected from H, C₁₋₁₈ alkyl or aryl; R⁷ is methyl, —CF₃, —N(R⁸)₂or —CH₂—N(R⁸)₂; R⁸ is independently selected from H or C₁₋₆ alkyl; R⁹ is(R⁸)₂, —R⁸—(CH₂)₂—N(R⁸)₂ or —R⁸—C(═NR⁸)[N(R⁸)₂]; and, R¹⁰ is H or C₁₋₄alkyl, wherein the siNA molecule is a single stranded antisensemolecule, or wherein the siNA molecule is a double-stranded moleculecomprising a sense strand that is complementary to the antisense strand;and wherein the single-stranded molecule is between 15 and 30nucleotides in length or wherein the antisense strand and the sensestrand are each independently 15 to 30 nucleotides in length.
 2. ThesiNA molecule of claim 1, wherein R³ is independently selected from H,hydroxy, F, Cl, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, aryl,

wherein R′ is selected from H, C₁₋₄ alkyl,

and R″ is selected from H, C₁₋₄ alkyl or aryl.
 3. The siNA molecule ofclaim 1, wherein A is —OCH₂—, —CH₂CH₂— or —CH═CH—.
 4. The siNA moleculeof claim 1, wherein D¹ and D^(1′) is independently selected fromhydroxyl, —OCH₃ or —OCH₂CH₃.
 5. The siNA molecule of claim 1, wherein Bis uracil, thymine, cytosine, 5-methylcytosine, adenine or guanine. 6.The siNA molecule of claim 1, wherein E is O.
 7. The siNA molecule ofclaim 1, wherein J is a phosphodiester internucleoside linking group ora phosphorothioate internucleoside linking group.
 8. The siNA moleculeof claim 1, wherein R¹ is H or hydroxyl, and R1′: is H, hydroxyl,halogen or —OR⁶.
 9. The siNA molecule of claim 1, wherein R¹, R^(1′) andR² are each H.
 10. The siNA molecule of claim 1, wherein the 5′ modifiednucleotide is


11. The siNA molecule claim 10, wherein B is thymine.
 12. The siNAmolecule of claim 1, wherein the siNA molecule is a single-stranded,antisense molecule.
 13. The siNA molecule of claim 12, wherein the siNAmolecule comprises one or more additional chemically-modifiednucleotides.
 14. The siNA molecule of claim 1, wherein the siNA moleculeis a double-stranded molecule comprising a sense strand that iscomplementary to the antisense strand.
 15. The siNA molecule of claim14, wherein the siNA molecule comprises one or more additionalchemically-modified nucleotides.
 16. A composition comprising the siNAmolecule of claim 1 in a pharmaceutically acceptable carrier or diluent.